High throughput methods of HLA typing

ABSTRACT

A method for determining an HLA genotype of a subject is disclosed. The method comprises (a) isolating template nucleic acid from the subject; (b) amplifying the template nucleic acid to generate sufficient product for each allele of at least one gene locus to be determined; (c) hybridizing the template nucleic acid with an immobilized array of capture oligonucleotides, each having a known nucleic acid sequence of an HLA allele; and (d) determining the particular capture oligonucleotide to which the template nucleic acid hybridizes, thereby determining the genotype of the subject. A number of additional methods that can eliminate or abbreviate additional steps are also described. Moreover, the present invention provides a method for determining tissue compatibility using the determined HLA genotype.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patentapplication Serial No. 60/172,768, filed on Dec. 20, 1999, the teachingsof which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] In general, this invention relates to typing and matching humanleukocyte antigens or alleles of human leukocyte antigens and inparticular, to high throughput screening methods of human leukocyteantigen matching or alleles of human leukocyte antigens.

BACKGROUND OF THE INVENTION

[0003] The human leukocyte antigen complex (also known as the majorhistocompatibility complex) spans approximately 3.5 million base pairson the short arm of chromosome 6. It is divisible into 3 separateregions which contain the class I, the class II and the class III genes.In humans, the class I HLA complex is about 2000 kb long and containsabout 20 genes. Within the class I region exist genes encoding the wellcharacterized class I MHC molecules designated HLA-A, HLA-B and HLA-C.In addition, there are nonclassical class I genes that include HLA-E,HLA-F, HLA-G, HLA-H, HLA-J and HLA-X as well as a new family known asMIC. The class II region contains three genes known as the HLA-DP,HLA-DQ and HLA-DR loci. These genes encode the α and β chains of theclassical class II MHC molecules designated HLA-DR, DP and DQ. Inhumans, nonclassical genes designated DM, DN and DO have also beenidentified within class II. The class III region contains aheterogeneous collection of more than 36 genes. Several completecomponents are encoded by three genes including TNF-α and TNF-β.

[0004] Any given copy of chromosome 6 can contain many differentalternative versions of each of the preceding genes and thus can yieldproteins with distinctly different sequences. The loci constituting theMHC are highly polymorphic, that is, many forms of the gene or allelesexist at each locus. Several hundred different allelic variants of classI and class II MHC molecules have been identified in humans. However,any one individual only expresses up to 6 different class I moleculesand up to 12 different class II, molecules.

[0005] The foregoing regions play a major role in determining whethertransplanted tissue will be accepted as self (histocompatible) orrejected as foreign (histoincompatible). For instance, within the classII region, three loci i.e., HLA-DR, DQ and DP are known to expressfunctional products. Pairs of A and B genes within these three lociencode heterodimeric protein products which are multi-allelic andalloreactive. In addition, combinations of epitopes on DR and/or DQmolecules are recognized by alloreactive T cells. This reactivity hasbeen used to define “Dw” types by cellular assays based upon the mixedlymphocyte reaction (MLR). It has been demonstrated that matching ofdonor and recipient HLA-DR and DQ alleles prior to allogeneictransplantation has an important influence on allograft survival.Therefore, HLA-DR and DQ matching is now generally undertaken as aclinical prerequisite for renal and bone marrow transplantation as wellas cord blood applications.

[0006] Until recently, matching has been confined to serological andcellular typing. For instance, in the microcytotoxicity test, whiteblood cells from the potential donor and recipient are distributed in amicrotiter plate and monoclonal antibodies specific for class I andclass II MHC alleles are added to different wells. Thereafter,complement is added to the wells and cytotoxicity is assessed by uptakeor exclusion to various dyes by the cells. If the white blood cellsexpress the MHC allele for a particular monoclonal antibody, then thecells will be lysed on addition of complement and these dead cells willtake up the dye. (see, Terasaki and McClelland, (1964) Nature, 204:998).However, serological typing is frequently problematic, due to theavailability and crossreactivity of alloantisera and because live cellsare required. A high degree of error and variability is also inherent inserological typing, which ultimately affects transplant outcome andsurvival (Sasazuki et al., (1998) New England J. of Medicine 339:1177-1185). Therefore, DNA typing is becoming more widely used as anadjunct, or alternative, to serological tests.

[0007] Initially, the most extensively employed DNA typing method forthe identification of these alleles has been restriction fragment lengthpolymorphism (RFLP) analysis. This well established method for HLA classII DNA typing suffers from a number of inherent drawbacks. RFLP typingis too time-consuming for clinical use prior to cadaveric renaltransplantation for example, and for this reason it is best suited tolive donor transplantation or retrospective studies. Furthermore, RFLPdoes not generally detect polymorphism within the exons which encodefunctionally significant HLA class II, epitopes, but relies upon thestrong linkage between alleles-specific nucleotide sequences withinthese exons and restriction endonuclease recognition site distributionwithin surrounding, generally noncoding, DNA.

[0008] In addition to restriction fragment length polymorphism(PCR-RFLP), an even more popular approach has been the hybridization ofPCR amplified products with sequence-specific oligonucleotide probes(PCR-SSO) to distinguish between HLA alleles (see, Tiercy et al., (1990)Blood Review 4: 9-15). This method requires a PCR product of the HLAlocus of interest be produced and then dotted onto nitrocellulosemembranes or strips. Then each membrane is hybridized with a sequencespecific probe, washed, and then analyzed by exposure to x-ray film orby colorimetric assay depending on the method of detection. Similar tothe PCR-SSP methodology, probes are made to the allelic polymorphic arearesponsible for the different HLA alleles. Each sample must behybridized and probed at least 100-200 different times for a completeClass I and II typing. Hybridization and detection methods for PCR-SSOtyping include the use of non-radioactive labeled probes, microplateformats, etc. (see e.g., Saiki et al. (1989) Proc. Natl. Acad. Sci.,U.S.A. 86: 6230-6234; Erlich et al. (1991) Eur. J Immunogenet. 18(1-2):33-55; Kawasaki et al. (1993) Methods Enzymol. 218:369-381), andautomated large scale HLA class II typing. A common drawback to thesemethods, however, is the relatively long assay times needed—generallyone to two days—and their relatively high complexity and resulting highcost. In addition, the necessity for sample transfers and washing stepsincreases the chances that small amounts of amplified DNA might becarried over between samples, creating the risk of false positives.

[0009] More recently, a molecular typing method using sequence specificprimer amplification (PCR-SSP) has been described (see, Olerup andZetterquist (1992) Tissue Antigens 39: 225-235). This PCR-SSP method issimple, useful and fast relative to PCR-SSO, since the detection step ismuch simpler. In PCR-SSP, allelic sequence specific primers amplify onlythe complementary template allele, allowing genetic variability to bedetected with a high degree of resolution. This method allowsdetermination of HLA type simply by whether or not amplificationproducts (collectively called an “amplicon”) are present or absentfollowing PCR. In PCR-SSP, detection of the amplification products isusually done by agarose gel electrophoresis followed by ethidium bromide(EtBr) staining of the gel. Unfortunately, the electrophoresis processtakes a long time and is not very suitable for large number of samples,which is a problem since each clinical sample requires testing for manypotential alleles. Gel electrophoresis also is not easily adapted forautomatic HLA-DNA typing.

[0010] Another HLA typing method is SSCP—Single-Stranded ConformationalPolymorphism. Briefly, single stranded PCR products of the different HLAloci are run on non-denaturing Polyacrylamide Gel Electrophoresis(PAGE). The single strands will migrate to a unique location based ontheir base pair composition. By comparison with known standards, atyping can be deduced. It is the only method that can determine truehomozygosity. However, many PAGE have to be run and many controls haveto be run to make it a viable typing method. This method is very timeconsuming, labor intensive, and not really suited for large volumeanalysis.

[0011] In view of the foregoing, what is needed in the art is a methodof determining genomic information from a highly polymorphic system suchas the HLA class I and class II regions. The present invention providesa highly accurate and efficient HLA class I and class II sequence-basedtyping method that is rapid, reliable and completely automatable.

SUMMARY OF THE INVENTION

[0012] The present invention provides new and improved methods for HLAtyping. In addition, the methods eliminate the reliance on agarose gelelectrophoresis usage for the sequence specific primer (SSP) method forperforming HLA DNA typing and obviates the reliance on using cumbersomeblot membranes for sequence-specific oligonucleotide probe hybridization(SSO) as well as many of the human errors associated with manualinterpretation of bands and assignment of alleles. Thus, the methods ofthe present invention decrease significantly the number of human errorsand the amount of time and effort it takes to perform DNA HLA typing.

[0013] In certain aspects, the present invention provides a method ofdetecting amplified DNA in which the risks of sample cross-contaminationand resulting false positive results are reduced. In addition, thepresent invention provides methods that can allow for reliable, rapidanalysis of multiple samples. Moreover, the present invention provides amethod of detecting amplified DNA that is relatively simple, and resultsin a relatively low cost per analysis and is amenable to automation andhigh throughput matching.

[0014] In one aspect, the present invention provides methods foridentifying an HLA genotype of a subject. The method involves (a)obtaining a sample containing a template nucleic acid from said subject;(b) amplifying the template nucleic acid with a plurality of HLAallele-specific forward primers and HLA allele-specific reverse primersto form amplification products, wherein the forward primers or reverseprimers comprise a detectable label; (c) hybridizing the amplificationproducts with a plurality of HLA locus-specific capture oligonucleotidesimmobilized on a solid phase to form a plurality of detectablecomplexes; and (d) detecting the detectable complexes to identify theHLA genotype of the subject.

[0015] Another aspect of the present invention provides methods foridentifying an HLA genotype of a subject that involves (a) obtaining asample containing a template nucleic acid from the subject; (b)amplifying the template nucleic acid with a plurality of HLAallele-specific forward primers and HLA allele-specific reverse primersto form amplification products, wherein the forward primers or reverseprimers contain a detectable label; (c) hybridizing the amplificationproducts with a plurality of HLA locus-specific capture oligonucleotidesto form a plurality of detectable complexes; (d) immobilizing thedetectable complexes on a solid phase; and (e) detecting the detectablecomplexes to identify the HLA genotype of the subject.

[0016] In yet another aspect of the invention, methods for identifyingan HLA genotype of a subject is provided that involves: immobilizing aplurality of HLA allele-specific reverse primers on a solid phase;amplifying the template nucleic acid with a plurality of HLAallele-specific forward primers and the immobilized reverse HLAallele-specific reverse primers to form amplification products; anddetecting the amplification products to identify the HLA genotype of thesubject.

[0017] In certain embodiments of the present invention, template nucleicacid that is isolated from blood or cord blood is amplified. Thetemplate nucleic acid can be any gene derived sequences, including, butnot limited to cDNA and genomic DNA.

[0018] In certain embodiments, oligonucleotides are immobilized on asolid phase. Examples of solid phase include, but are not limited to: abead, a chip, a microtiter plate, a polycarbonate microtiter plate,polystyrene microtiter plate, and a slide. The methods of the presentinvention can be also used to determine class I and class II HLAgenotypes. In certain embodiments, HLA allele-specific forward primersand HLA allele-specific reverse primers are used to amplify the templatenucleic acid to generate amplification products. In some embodiments,the HLA allele-specific primers are selected from primers denoted as SEQID NOS:1-160 and SEQ ID NOS: 169-269.

[0019] In some embodiments of the invention, capture oligonucleotidesare employed. In certain preferred embodiments, locus-specific captureoligonucleotides are used in the HLA genotyping methods and can beselected from the primers such as SEQ ID NOS: 272-277 and SEQ IDNOS:165-168. The capture oligonucleotides can be modified with a moietythat aids in immobilizing the capture oligonucleotide to a solid phase.In certain embodiments, moieties such as a 5′ amine group or a 5′(T)₅₋₂₀oligonucleotide sequence are utilized.

[0020] Detectable labels can be used with certain embodiments of thepresent invention. Examples of a detectable label, include, but are notlimited to a radioactive moiety, a fluorescent moiety, achemiluminescent moiety, an antigen, or a binding protein. In certainembodiments, fluorescent moieties such as fluorescein or5-(2′-aminoethyl) aminonaphtalene-1-sulfonic acid (EDANS) are attachedto oligonucleotides to facilitate detection.

[0021] These embodiments as well as additional objects and advantageswill become more readily apparent when read with the accompanying figureand detailed description which follows.

DEFINITIONS

[0022] An “allele” is one of the different nucleic acid sequences of agene at a particular locus on a chromosome. One or more geneticdifferences can constitute an allele. Examples of HLA allele sequencesare set out in Mason and Parham (1998) Tissue Antigens 51: 417-66, whichlist HLA-A, HLA-B, and HLA-C alleles and Marsh et al. (1992) Hum.Immunol. 35:1, which list HLA Class II alleles for DRA, DRB, DQA1, DQB1,DPA1, and DPB1.

[0023] A “locus” is a discrete location on a chromosome that constitutesa gene. Exemplary loci are the class I MHC genes designated HLA-A, HLA-Band HLA-C; nonclassical class I genes including HLA-E, HLA-F, HLA-G,HLA-H, HLA-J and HLA-X, MIC; and class II genes such as HLA-DP, HLA-DQand HLA-DR.

[0024] A method of “identifying an HLA genotype” is a method thatpermits the determination or assignment of one or more geneticallydistinct HLA genetic polymorphisms.

[0025] The term “amplifying” refers to a reaction wherein the templatenucleic acid, or portions thereof, are duplicated at least once. Unlessspecifically stated “amplifying” may refer to arithmetic, logarithmic,or exponential amplification. The amplification of a nucleic acid cantake place using any nucleic acid amplification system, both isothermaland thermal gradient based, including but not limited to, polymerasechain reaction (PCR), reverse-transcription-polymerase chain reaction(RT-PCR), ligase chain reaction (LCR), self-sustained sequence reaction(3 SR), and transcription mediated amplifications (TMA). Typical nucleicacid amplification mixtures (e.g. PCR reaction mixture) include anucleic acid template that is to be amplified, a nucleic acidpolymerase, nucleic acid primer sequence(s), and nucleotidetriphosphates, and a buffer containing all of the ion species requiredfor the amplification reaction.

[0026] An “amplification product” is a single stranded or doublestranded DNA or RNA or any other nucleic acid products of isothermal andthermal gradient amplification reactions that include PCR, TMA, 3SR,LCR, etc.

[0027] The phrase “template nucleic acid” refers to a nucleic acidpolymer that is sought to be copied or amplified. The “template nucleicacid(s)” can be isolated or purified from a cell, tissue, animal, etc.Alternatively, the “template nucleic acid(s)” can be contained in alysate of a cell, tissue, animal, etc. The template nucleic acid cancontain genomic DNA, cDNA, plasmid DNA, etc.

[0028] An “HLA allele-specific” primer is an oligonucleotide thathybridizes to nucleic acid sequence variations that define or partiallydefine that particular HLA allele.

[0029] An “HLA locus-specific” primer is an oligonucleotide that permitsthe amplification of a HLA locus sequence or that can hybridizespecifically to an HLA locus.

[0030] A “forward primer” and a “reverse primer” constitute a pair ofprimers that can bind to a template nucleic acid and under properamplification conditions produce an amplification product. If theforward primer is binding to the sense strand then the reverse primer isbinding to antisense strand. Alternatively, if the forward primer isbinding to the antisense strand then the reverse primer is binding tosense strand. In essence, the forward or reverse primer can bind toeither strand as long as the other reverse or forward primer binds tothe opposite strand.

[0031] The term “detectable label” refers to a moiety that is attachedthrough covalent or non-covalent means to an oligonucleotide. A“detectable label” can be a radioactive moiety, a fluorescent moiety, achemiluminescent moiety, etc.

[0032] The term “fluorescent label” refers to label that accepts radiantenergy of one wavelength and emits radiant energy of a secondwavelength.

[0033] The phrase “hybridizing” refers to the binding, duplexing, orhybridizing of a molecule only to a particular nucleotide sequence orsubsequence through specific binding of two nucleic acids throughcomplementary base pairing. Hybridization typically involves theformation of hydrogen bonds between nucleotides in one nucleic acid andcomplementary sequences in the second nucleic acid.

[0034] The phrase “hybridizing specifically” refers to hybridizing thatis carried out under stringent conditions.

[0035] The term “stringent conditions” refers to conditions under whicha capture oligonucleotide, oligonucleotide or amplification product willhybridize to its target subsequence, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. Generally, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength, pH, and nucleic acid concentration) atwhich 50% of the probes complementary to the target sequence hybridizeto the target sequence at equilibrium. (As the target sequences aregenerally present in excess, at Tm, 50% of the capture oligonucleotidesare occupied at equilibrium). Typically, stringent conditions will bethose in which the salt concentration is at most about 0.01 to 1.0 M Na⁺ion concentration (or other salts) at pH 7.0 to 8.3 and the temperatureis at least about 30° C. for short probes (e.g., 10 to 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. An extensive guide to thehybridization and washing of nucleic acids is found in Tijssen (1993)Laboratory Techniques in biochemistry and molecularbiology—hybridization with nucleic acid probes parts I and II, Elsevier,N.Y., and, Choo (ed) (1994) Methods In Molecular Biology Volume 33-InSitu Hybridization Protocols Humana Press Inc., New Jersey; Sambrook etal., Molecular Cloning, A Laboratory Manual (2^(nd) ed. 1989); CurrentProtocols in Molecular Biology (Ausubel et al., eds., (1994)).

[0036] The term “complementary base pair” refers to a pair of bases(nucleotides) each in a separate nucleic acid in which each base of thepair is hydrogen bonded to the other. A “classical” (Watson-Crick) basepair always contains one purine and one pyrimidine; adenine pairsspecifically with thymine (A-T), guanine with cytosine (G-C), uracilwith adenine (U-A). The two bases in a classical base pair are said tobe complementary to each other.

[0037] “Bind(s) substantially” refers to complementary hybridizationbetween a capture nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

[0038] The term “capture oligonucleotide” refers to a nucleic acidsequence or nucleic acid subsequence that can hybridize to anotheroligonucleotide, amplification product, etc. and has the ability to beimmobilized to a solid phase. A capture oligonucleotide typicallyhybridizes to at least a portion of an amplification product containingcomplementary sequences under stringent conditions.

[0039] A “HLA locus-specific capture oligonucleotide” is a captureoligonucleotide that is complementary to and hybridizes to a conservedregion of an HLA locus. For example a “HLA locus-specific captureoligonucleotide” that is specific for the HLA-A locus will hybridize toone or more conserved regions or subsequences of the HLA-A locus.

[0040] A compound is “immobilized on a solid phase” when it is directlyor indirectly attached to the solid phase. Such immobilization may bethrough covalent and/or non-covalent bonds.

[0041] The term “corresponding nucleotide, ” is used to refer to theposition of a nucleotide in a first nucleic acid by reference to asecond nucleic acid. Thus, a corresponding nucleotide refers to anucleotide that it is positionally located opposite to a base whereneighboring bases are all hybridized pairs.

[0042] “Subsequence” refers to a sequence of nucleic acids that comprisea part of a longer sequence of nucleic acids.

[0043] The term “portions” should similarly be viewed broadly, and wouldinclude the case where a “portion” of a DNA strand is in fact the entirestrand.

[0044] The term “specificity” refers to the proportion of negative testresults that are true negative test result. Negative test resultsinclude false positives and true negative test results.

[0045] The term “sensitivity” is meant to refer to the ability of ananalytical method to detect small amounts of analyte. Thus, as usedhere, a more sensitive method for the detection of amplified DNA, forexample, would be better able to detect small amounts of such DNA thanwould a less sensitive method. “Sensitivity” refers to the proportion ofexpected results that have a positive test result.

[0046] The term “reproducibility” as used herein refers to the generalability of an analytical procedure to give the same result when carriedout repeatedly on aliquots of the same sample.

[0047] The term “amplicon” is used herein to mean a population of DNAmolecules that has been produced by amplification, e.g., by PCR.

[0048] The term “molecular beacon,” as used herein refers to a moleculecapable of participating in a specific binding reaction and whosefluorescence activity changes when the molecule participates in thatbinding reaction.

DETAILED DESCRIPTION

[0049] I. Introduction

[0050] The present invention provides methods for HLA genotyping ofhuman leukocyte antigens, as well as other molecular diagnosticprotocols relating to the detection of DNA sequences and sequencevariations using nucleic acid amplification methods. Advantageously, themethods described herein can be used to detect genetic mutations, detectcancer gene mutations, microbial and cancer drug resistance mutations,detection of viruses, bacteria, fungi, parasites and any other microbes,forensics, parentage, etc.

[0051] In particular, the methods of the present invention are usefulfor determining HLA genotypes of samples from subjects. Such genotypingis important in the clinical arena for the diagnosis of disease,transplantation of organs, and bone marrow and cord blood applications.

[0052] In the present invention, allelic-specific HLA primers are usedto amplify HLA sequences. In some embodiments, these amplificationproducts can be immobilized to a solid phase using a locus-specific oran allele-specific capture oligonucleotide. In certain embodiments, thelocus-specific capture oligonucleotides are preferred as fewer captureoligonucleotides need to be generated to carry out the HLA genotyping.In other embodiments, one HLA-specific primer is immobilized to a solidphase and the target is amplified using another HLA-specific primer thatis free in solution. The advantages and details for carrying out thepresent invention will be discussed more fully below.

[0053] II. Materials Used in the Present Invention

[0054] Oligonucleotides

[0055] Oligonucleotides used in the present invention (e.g., allele andlocus-specific oligonucleotides) can be chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage & Caruthers, (1981) Tetrahedron Letts. 22:1859-1862, using anautomated synthesizer, as described in Van Devanter et al., (1984)Nucleic Acids Res. 12: 6159-6168. Purification of oligonucleotides istypically by either native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson & Reanier, (1983) J. Chrom.255:137-149.

[0056] HLA Allele-Specific Primers

[0057] The HLA allele-specific primers used in the present invention aredesigned to amplify HLA allele sequences. Since 1995, 213 class I(HLA-A, HLA-B, and HLA-C) and 256 class II (HLA-DR, HLA-DP, and HLA-DQ)alleles had been identified and sequenced (see e.g., Krausa and Browning(1996) Detection of HLA gene polymorphism in Browning M, McMichael A,ed. HLA and MHC: genes, molecules and function. Oxford: Bios ScientificPublishers Limited, pp. 113-138), with new alleles being discovered allthe time. The sequences of many of these alleles are publicly availablethrough GenBank and other gene databases and have been published (seee.g., Mason and Parham (1998) Tissue Antigens 51: 417-66, listing HLA-A,HLA-B, and HLA-C alleles; Marsh et al. (1992) Hum. Immunol. 35:1,listing HLA Class II alleles-DRA, DRB, DQA1, DQB1, DPA1, and DPB1).Also, the use of allele-specific primers (sequence-specific primers(SSP)) has permitted the specific amplification of HLA allele sequences(see e.g., Bunce and Welsh (1994) Tissue Antigens 43: 7-17,amplification of HLA-C alleles; Bunce et al. (1995) Tissue Antigens 46:355-67, amplification of HLA-A.B.C. DRB 1, DRB3, DRB4, DRB5 & DQB1alleles with sequence-specific primers; Gilchrist et al. (1998) TissueAntigens 51: 51-61, HLA-DP typing with sequence specific primers).

[0058] In the design of the HLA primer pairs for the primer mixes,primers were selected based on the published HLA sequences available inthe literature. A chart of the HLA alleles and sequences was examinedand the polymorphic sites were identified. Then pairs of primers wereselected that would produce PCR products to a group of HLA alleles. Thesequence specific nucleic acid amplification reaction typically uses atleast a pair of PCR primers for each allele, both of which contain thediscriminating sequences with at least one or more of the changednucleotides at the 3′ end of each PCR primer. Since the 3′ end is theend where polymerization takes place, if a mismatch occurs due tosequence non-complementarily, nucleic acid amplification will take placeand one would not expect a “false positive.” However, if a match occurs,then the amplification can proceed. For example, HLA class Iallele-specific primers and HLA class II allele-specific primers arelisted in Table 1 (SEQ ID NOS: 1-160) and 2 (SEQ ID NOS: 169-269),respectively. Examples of control primers listed in Table 1 are CI53(SEQ ID NO: 161), CI54 (SEQ ID NO: 162), CI148 (SEQ ID NO: 163), andCI149 (SEQ ID NO: 164). Examples of control primers listed in Table 2are DPA-E(PC) (SEQ ID NO: 270), and DPA-F (PC) (SEQ ID NO: 271). TheClass I primers are selected to amplify Class I exon 2 and exon 3products. The Class II primers are selected to amplify Class II exon 2products. In certain embodiments, the primers listed in Tables 3 and 4are used as exemplary groups of primer pairs and the HLA specificitiesthese pairs can identify after successful positive PCR amplificationswith the appropriate DNA templates for HLA class I and II allelesrespectively. By utilizing a pair of primers, each PCR reactionidentifies two sites of polymorphism and therefore increases thespecificity of the reaction. Those of skill in the art will recognize amultitude of oligonucleotide compositions that can be used as HLAallele-specific primers to specifically amplify HLA allele sequences. Inaddition, customized sets of HLA-specific primers can be created tocater to detection of the allele distribution for various ethnicities orracial groups by simply changing the primer pair combinations. In thismanner, detection of new alleles can be easily added to the methods ofthe present invention.

[0059] Capture Oligonucleotides

[0060] In certain embodiments, the invention involves locus-specificcapture oligonucleotides or allele-specific capture oligonucleotides.Locus-specific oligonucleotide can hybridize to a conserved region in aHLA locus; a locus-specific capture oligonucleotide has the ability tohybridize to some or all of the sequences that can be generated by theamplification of HLA allele sequences using HLA-specific primers.Locus-specific sequences have been identified in HLA loci. For example,locus-specific sequences for HLA-class I genes have been delineated inthe first and third introns flanking the polymorphic second and thirdexons (see e.g., Cereb et al. (1995) Tissue Antigens 45: 1-11). Thecapture oligonucleotides should be of such length and composition so asto be able to hybridize with the allele-specific PCR products. Incertain embodiments, HLA locus-specific class I capture oligonucleotidescontain the flowing sequences: for HLA-A (CICptA1, Class I Capture OligoA1, 5′CGCCTACGACGGCAAGGATTACATCGCCC3′(SEQ ID NO:165); and CICptA2, ClassI Capture Oligo A2, 5′GATGGAGCCGCGGTGGATAGAGCAGGAGGG3′(SEQ ID NO:166),for HLA-B (CICptB1, Class I Capture Oligo B1,5′CAGTTCGTGAGGTTCGACAGCGACGCC3′(SEQ ID NO:167), and CICptB2, Class ICapture Oligo B2, 5′CTGCGCGGCTACTACAACCAGAGCGAGGCC3′(SEQ ID NO:168). Inother embodiments, HLA locus-specific class II capture oligonucleotidescontain the following sequences:

[0061] for HLA-DQ (DQCPT1, 5′CACGTCGCTGTCGAAGCGCACGTACTCCTC3′(SEQ IDNO:272); DQCPT2, 5′CACGTCGCTGTCGAAGCGGACGATCTCCTT3′(SEQ ID NO:273);DQCPT3, 5′CACGTCGCTGTCGAAGCGTGCGTACTCCTC3′(SEQ ID NO:274); DQCPT4,5′CACGTCGCTGTCGAAGCGCGCGTACTCCTC3′(SEQ ID NO:275); and

[0062] DQCPT5, 5′CACGTCGCTGTCGAAGCGCACGTCCTCCTC3′(SEQ ID NO:276), forHLA-DR (DRCPT1, DRCP, 5′TGGCGTGGGCGAGGCAGGGTAACTTCTTTA3′(SEQ IDNO:277)). In certain embodiments, it may require the use of more thanone capture oligonucleotide to hybridize to all of the HLA alleleamplification products.

[0063] Modification of Oligonucleotides

[0064] In certain embodiments of the present invention, oligonucleotidesare modified or synthesized as modified oligonucleotides to facilitateimmobilization or detection.

[0065] Immobilization Modifications

[0066] In certain embodiments, where capture oligonucleotides are usedor where an immobilized amplification primer is used, it is desirable tomodify the particular oligonucleotide to affix it to a solid phase orsupport. It is desired that the modification of the captureoligonucleotide does not interfere with its ability to bind to an HLAallele-specific amplification product. Those of skill in the art willrecognize a variety of methods to immobilize oligonucleotides to a solidphase. For example, oligonucleotides can be directly or indirectlyimmobilized on a solid phase. The oligonucleotides can be immobilizeddirectly to the solid phase through covalent and non-covalent bonds. Forexample, the 5′ end of an oligonucleotide can be synthesized with anamine moiety (see Kawasaki et al. (1993)). In certain embodiments, anamine moiety with a C6 carbon spacer is conjugated to the 5′ end of acapture oligo or amplification primer. The amine-modified primers areaffixed to the surface of a substrate such a Biodyne C membrane (PallBiosupport) (Kawasaki et al. (1993)) or through a commercially availablemicrotiter plate (e.g., Xenobind™ (Covalent Binding Microwell Plates),Xenopore, Hawthorne, N.J.). Alternatively a polythymidine (polyT)stretch can be added to an oligonucleotide by terminaldeoxyribonucletotidyltransferase (Saiki et al. (1989)). Such a polyTstretch can be fixed to many solid substrates (e.g., nylon) using UVlight leaving the rest of the oligonucleotide free to hybridize toanother nucleic acid. Preferably, the polyT stretch is from 5 to 20 T's.

[0067] Alternatively, the oligonucleotides can be indirectly bound tothe solid phase by coating the solid phase with a substance or moleculethat can bind to the oligonucleotides. Biotinylated oligonucleotides canalso be used as capture oligonucleotides. Methods are known in the artfor synthesizing biotinylated oligonucleotide (e.g., by synthesizing aprimer with a biotinylated 5′ end nucleotide as the terminal residue)(see e.g., Innis et al. (1990)). Biotinylated oligonucleotides can beaffixed to a substrate that is coated with avidin.

[0068] A high density array of capture oligonucleotides or amplificationprimers can be also synthesized on a substrate by attachingphotoremovable groups to the surface of a substrate, exposing selectedregions of the substrate to light to activate those regions, attaching anucleic acid monomer with a photoremovable group to the activatedregions, and repeating the steps of activation and attachment untilprobes of the desired length and sequences are synthesized. (See, e.g.,Fodor et al. (1991) Science 251: 767-773 and U.S. Pat. No. 5,143,854).The resulting array of oligonucleotides can then be used to in themethods of the present invention.

[0069] A variety of solid supports or phases can be used in the presentinvention. Examples of solid supports include, without limitation, bead,microtiter plates, and chips. Beads can be composed of materials such asSepharose, agarose, polystyrene, etc. and can be paramagnetic.Microtiter plates are commercially available in a variety of formats(e.g., 96, 384 and 1536 well plates) and materials (e.g., polystyrene).The plates can be either polycarbonate plates in which case the thermalgradient nucleic acid amplification reaction (such as PCR) can happendirectly in the well or polystyrene in which case the thermal gradientnucleic acid amplification reaction (such as PCR) has to take place in aseparate polycarbonate plate and transferred to the surface modified andoligonucleotide attached plate. Isothermal nucleic acid amplificationmethods can be conducted in polystyrene plates. chips can be comprisedof a variety of materials, layers and substrates. Polymers which may beused as solid supports or phases include, but are not limited to, thefollowing: polystyrene; poly(tetra)fluoroethylene (PTFE);polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;polyvinylethylene; polyethyleneimine; poly(etherether)ketone;polyoxymethylene (POM); polyvinylphenol; polylactides;polymethacrylimide (PMI); polyatkenesulfone (PAS); polypropylene;polyethylene; polyhydroxyethylmethacrylate HEMA); polydimethylsiloxane;polyacrylamide; polyimide; and block-copolymers. The solid support onwhich an oligonucleotide resides may also be a combination of any of theaforementioned solid support materials.

[0070] Oligonucleotides Containing Detectable Labels

[0071] Detectable labels can also be attached to oligonucleotides tofacilitate detection of the oligonucleotide in an analyte. Detectablelabels can be detected either directly or indirectly, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radiolabels (e.g., ³H, ¹³C, ¹⁴C, ³²P,³⁵S, 125I, etc.), fluorescent dyes, fluorophores, fluorescent moieties,chemiluminescent moieties, electron-dense reagents, enzymes and theirsubstrates (e.g., as commonly used in enzyme-linked immunoassays, e.g.,alkaline phosphatase and horseradish peroxidase), biotin-streptavidin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available. The label or detectable moiety is typicallybound, either covalently, through a linker or chemical bound, or throughionic, van der Waals or hydrogen bonds to the molecule to be detected.

[0072] The detectable label should be stable to the amplificationsconditions used and should permit direct or indirect detection. Indirectdetection often involves the presence of one or more detection reagents.For example, one detectable label, biotin can be detected using anavidin conjugate such as avidin conjugated to an enzyme such asperoxidase (e.g., HRP), and a calorimetric substrate for peroxidase(e.g., TMB). The formation of calorimetric product can easily bedetected using a spectrophotometer. For example, in certain embodiments,the primers listed in Tables 5 and 6 are biotinylated.

[0073] In certain embodiments, oligonucleotides comprising a quencherand a fluorophore moiety (molecular beacons) are contemplated. MolecularBeacons are single stranded oligonucleotide probes designed to havehairpin configuration by virtue of the presence of five to sevencomplementary nucleotides at their termini. The loop portion (10-40nucleotides) is chosen so that the probe-amplification product hybrid isstable at the annealing temperature. The length of the arm sequences(5-7 nucleotides) is chosen so that a stem is formed at the annealingtemperature of the polymerase chain reaction. Also the stem or armsequence must be designed to ensure that the two arms hybridize to eachother but not to the probe sequence. One end would carry a fluorophore(e.g. 5-(2′-aminoethyl) aminonaphtalene-1-sulfonic acid (EDANS) and theother a quencher (e.g. 4-(4′-dimethylaminophenylazo)benzoic acid(DABCYL). When a probe is not hybridized to its complementary targetsequence, the hairpin folding reaction would take place and fluorescencedoes not occur due to quenching. Quenching occurs because the energygiven off as light during fluorescence is transferred to the quencherand dissipated as heat. Since the energy is released as heat instead oflight, the fluorescence is said to be quenched. However, if acomplementary target sequence is present, hybridization to the targetsequence would be favored over the internal hairpin due to the increasedstability as a result of the longer stretches of complementary sequence.The hairpin would open up thus allowing for release of quenching and theprobes to fluoresce. In the fluorophore-quencher pair example givenabove, when stimulated by UV light with a peak wavelength of 336 nm,EDANS emits a brilliant blue fluorescence with a peak wavelength of 490nm. (Tyagi et al., (1996) Nature Biotechnology 14: 303-308; Tyagi et al.(1998) Nature Biotechnology 16:49-53; Paitek et al. (1998) NatureBiotechnology 16: 359-63; Kostrikis et al. (1998) Science279:1228-1229).

[0074] III. Source of HLA Gene Sequences

[0075] The template HLA DNA sequences are contained in samplescontaining nucleic acid (e.g., DNA, RNA, etc.), which are obtained froma biological source. In certain embodiments, the nucleic acid isisolated from a biological source containing HLA gene sequences. Thenucleic acid may be from any species having HLA gene sequences, whichinclude but are not limited to, a human, a chimpanzee, a simian, amouse, etc. Methods are known for lysing biological samples andpreparing extracts or purifying DNA, RNA, etc. See, Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)). In some embodiments,the biological source is blood, and is more preferably cord blood (e.g.,blood from an umbilical cord). In methods involving cord blood or blood,two isolation procedures are preferred: Salt extraction with ethanolprecipitation; and the Qiagen QIAamp® isolation method. For the saltextraction method, the cells are first lysed and centrifuged. Then wateris added and the sample is centrifuged again. The pellet is digestedwith Proteinase K. The DNA is then extracted by the addition of 6MGuanidine HCl and incubation at 70° C. for several minutes. The sampleis centrifuged again and the supernatant is precipitated with cold 95%Ethanol. The pellet is then dried and resuspended in the appropriatebuffer.

[0076] RNA template sequences that are amplified using the methods andcompositions of the present invention may be a single RNA template ordifferent RNA templates. The RNA can be isolated as total RNA from acell, bacterium, virus etc. See, Ausubel et al. The total RNA may besubsequently purified as poly A+RNA or purified in a different manner toisolate certain species of interest. See Ausubel et al. Alternatively,the template RNA can be transcribed in vitro and used in the presentinvention. The RNA template sequence could also be reverse transcribedinto cDNA and used as a nucleic acid template in the methods of thepresent invention.

[0077] IV. Amplification of HLA Gene Sequences From Nucleic Acid

[0078] The methods of the present invention involve the direct orindirect detection of HLA gene sequences that have been amplified fromDNA or reverse transcribed DNA. To amplify the desired nucleic acid forHLA gene sequences, the following are usually present in the reactionvessel: template nucleic acid, nucleic acid polymerase, a molar excessof dNTPs, an antisense primer(s), and a sense-primer(s), for copying aHLA gene sequence from a template nucleic acid. Preferably, the reactioncan be carried out in a thermal cycler oven to facilitate incubationtimes at the desired temperatures.

[0079] Reaction Components

[0080] Oligonucleotide Primers

[0081] The oligonucleotides that are used in the present invention, aswell as oligonucleotides designed to detect amplification products, canbe chemically synthesized as described above. These oligonucleotides canbe labeled with radioisotopes, chemiluminescent moieties, or fluorescentmoieties, etc. in a covalent or non-covalent manner. Such labels areuseful for the characterization and detection of amplification productsusing the methods and compositions of the present invention.

[0082] Buffer

[0083] Buffers that may be employed are borate, phosphate, carbonate,barbital, Tris, etc. based buffers. See Rose et al., U.S. Pat. No.5,508,178. The pH of the reaction should be maintained in the range ofabout 4.5 to about 9.5. See U.S. Pat. No. 5,508,178. The standard bufferused in amplification reactions is a Tris based buffer between 10 and 50mM with a pH of around 8.3 to 8.8. See Innis et al. (1990). In certainembodiments of the invention, a preferred buffer for the presentinvention is 150 mM Tris-HCl pH 8.8 for the amplification of class-I HLAsequences and 20 mM Tris HCl pH 8.8 for class II HLA sequences.

[0084] Salt Concentration

[0085] The concentration of salt present in the reaction can affect theability of primers to anneal to the template nucleic acid. See Innis etal. (1990). For example, potassium chloride can be added up to aconcentration of about 50 mM to the reaction mixture to promote primerannealing. Sodium chloride can also be added to promote primerannealing. See Innis et al. (1990). In certain embodiments of theinvention, the preferred salts are 30 mM Ammonium Chloride for class IHLA sequences and 100 mM KC1 for class II sequences.

[0086] Magnesium Ion Concentration

[0087] The concentration of magnesium ion in the reaction can becritical to amplifying the desired sequence(s). See Innis et al. (1990).Primer annealing, strand denaturation, amplification specificity,primer-dimer formation, and enzyme activity are all examples ofparameters that are affected by magnesium concentration. See Innis etal. (1990). Amplification reactions should contain about a 0.5 to abouta 5 mM magnesium concentration excess over the concentration of dNTPs.The presence of magnesium chelators in the reaction can affect theoptimal magnesium concentration. A series of amplification reactions canbe carried out over a range of magnesium concentrations to determine theoptimal magnesium concentration. The optimal magnesium concentration canvary depending on the nature of the template nucleic acid(s) and theprimers being used, among other parameters. In certain embodiments ofthe invention, the preferred magnesium concentrations are 4 mM MgCl₂ and3.4 MM MgCl₂, for class I HLA sequences and class II HLA sequences,respectively.

[0088] Deoxyribonucleotide Triphosphate Concentration

[0089] Deoxyribonucleotide triphosphates (dNTPs) are added to thereaction to a final concentration of about 20 μM to about 300 μM. Eachof the four dNTPs (G, A, C, T) should be present at equivalentconcentrations. See Innis et al. In certain embodiments, 166 μM dNTP isthe preferred concentration of nucleotides.

[0090] Nucleic Acid Polymerase

[0091] A variety of DNA dependent polymerases are commercially availablethat will function using the methods and compositions of the presentinvention. For example, Taq DNA Polymerase may be used to amplifytemplate DNA sequences. Also, a reverse transcriptase can be used incertain embodiments of the present invention. Reverse transcriptases,such as the thermostable C. therm polymerase from Roche, are also widelyavailable on a commercial basis.

[0092] Other Agents

[0093] Assorted other agents or compounds are sometime added to thereaction to achieve the desired results. For example, DMSO can be addedto the reaction, but is reported to inhibit the activity of Taq DNAPolymerase. However, DMSO has been recommended for the amplification ofmultiple template sequences in the same reaction. See Innis et al.Stabilizing agents such as gelatin, bovine serum albumin, and non-ionicdetergents (e.g. Tween-20) are commonly added to amplificationreactions. See Innis et al. For the amplification of class II sequences,the addition of 0.2% Triton X-100 has been found to be preferred. Inaddition, to enhance specificity by decreasing spurious priming, methodsthat incorporate “hot start” (e.g., AmpliWax®) (Applied Biosystems,Inc.), or an monoclonal antibody to Taq polymerase (CLONTECHLaboratories, Inc.) can be used to increase the specificity of anamplification reaction.

[0094] Amplification Programs

[0095] To amplify the HLA gene sequences of interest, the amplificationreaction mixture is subjected to a series of temperatures to repeatedlydenature the nucleic acid, anneal the oligonucleotide primers, andextend the primers with the polymerase. The use of a thermal cyclingdevice can greatly facilitate the temperature cycling required incertain embodiments of the present invention. The optimum denaturing,annealing and extending temperatures can be determined by one of skillin the art for a particular oligonucleotide primer pair(s) and HLA genetemplate(s). In general, the extension step is carried out at atemperature of about 72° C. and the denaturing step is carried out atabout 96° C. In addition, it may be necessary to carry out differentsets of amplification cycles in succession to achieve the desiredresults. In addition, the number of cycles is an importantconsideration. Typically, one of skill in the art can carry outexperiments to determine what is the optimum number of cycles to amplifythe desired template(s).

[0096] The annealing temperature is of critical importance in anyamplification reaction. If the annealing temperature is too low,non-specific amplification of undesired templates can arise. If theannealing temperature is too high, the template may not be efficientlyamplified if at all. Determining the optimum annealing temperature forin reactions that involve large numbers of different oligonucleotidesequences and HLA templates is particularly important. A preferredamplification program for amplifying template HLA gene sequences whereboth primers are in solution is the following 6-stage program: 1.)  1Cycle 97° C. for 20 seconds 2.)  5 Cycles 97° C. for 35 seconds, 61° C.for 45 seconds, 72° C. for 40 seconds 3.) 25 Cycles 97° C. for 20seconds, 59° C. for 45 seconds, 72° C. for 40 seconds 4.)  4 Cycles 97°C. for 20 seconds, 57° C. for 45 seconds, 72° C. for 90 seconds 5.)  1Cycle 72° C. for 4 minutes 6.)  1 Cycle 30° C. for 1 second.

[0097] A number of controls can be used in the amplification methodsdescribed herein. They include, but are not limited to: 1. Omission ofPrimers—Control of spurious priming; 2. Known negative control—Controlof specificity; 3. Known positive control—Control of sensitivity; 4.Omission of DNA Polymerase—Detection of non-specific probe and/orenzyme/antibody sticking; 5. Use of irrelevant probes forhybridization—Control for hybridization; 6. Amplification of endogenouscontrol DNA sequence—Detection of false negatives, control of DNA/RNAquality.

[0098] V. Washing

[0099] After a hybridization step or after solid-phase PCR (e.g.,amplification with an immobilized primer), a solid phase can be washedwith a buffer to decrease non-specific binding, to wash away unboundprimers, or to provide a solution that is more appropriate forsubsequent detection of a detectable label, etc. Where anoligonucleotide has been immobilized or hybridized to an oligonucleotideon a solid support, the unbound oligonucleotides can be washed from abound complex using variety of separation methods known in the art.There are many separation methods known in the art (e.g., filtering,sedimenting, centrifuging, decanting, precipitation, etc.) that can beused or adapted for use in the present invention. For example, where theamplification product is immobilized on a microtiter plate, the unboundoligonucleotides can be aspirated from the well, leaving behind thoseamplification products, HLA allele sequences, etc. that are bound to asolid phase. Another separation method is the immobilization of anamplification product, HLA allele sequence, etc. on a paramagnetic bead,and the decantation or aspiration of the unbound primers andoligonucleotides leaving behind the bound complex containing adetectable label remaining on the solid phase. Commercial kits, methodsand systems are commercially available and can be adapted or used withthe present invention (e.g., the KingFisher™ system from ThermoLabsystems, Inc.).

[0100] A wash buffer can contain a detergent, or other agents, andcompositions that are compatible with retention of the bound complex onthe solid phase. A blocking agent is generally present in the washbuffer. Blocking agents include, but are not limited to non-fat drymilk, herring sperm DNA, dextran sulfate, and BSA. For example, a washbuffer that can be used in the present invention is a solution of 0.1%BSA in PBS. The use of 0.1% BSA results in optimum results. One or morewashes may be necessary to achieve optimum lowering of non-specificbinding.

[0101] VI. Detection

[0102] A wide range of methods can be used to detect the presence ofoligonucleotides that contain a detectable label. The method ofdetection depends on the nature of the detectable label that is present.If the label is directly or indirectly capable of generating a signal inthe visible light range, then a spectrophotometer can be used.Similarly, a fluorescent detectable label or signal generated therefromcan be measured using a fluorescent spectrophotometer. Alternatively,luminometers can be used to measure chemiluminescent signals. Isotopiclabels can be measured using a liquid scintillation counter or in somecases-x-ray film. In certain embodiments, it is preferred to use aspectrophotometric plate reader that can read microtiter plates in anautomated system.

[0103] VII. Analysis of Results of Assays

[0104] Computer programs containing algorithm(s) can be used to score,interpret and assign HLA alleles in certain embodiments of the presentinvention. Briefly, the data from a detection instrument (e.g., aspectrophotometer, an ELISA reader, a scintillation counter, etc.) canbe analyzed through the use of a computer program that compares thevalues of each sample against a reference value(s).

[0105] For example, computer programs for the ELISA format readers takereadings below a designated threshold and label such as negative andvalues above the same thresholds as positives. A positive well or acombination of certain wells would then represent a specific genesequence or allele and be scored as such with the automated program. Theoptical density (O.D.) values obtained from reading of the wells of theELISA plate readers are given as numerical values ranging from0.000-2.000. This information is automatically downloaded onto theattached computers via the vendor provided software. The O.D. values aresaved in a spreadsheet format in the vendor provided program as rawdata.

[0106] The first step in computer analysis of the data is to validateand assign the negative control reading from the negative control well,which always exists in the same well location on the plate. A properlyperformed negative control is assigned as the negative value. In someembodiments where peroxidase is used with TMB, negative controls aredeemed properly performed when the O.D. values are below 0.2. The usualO.D. values of a negative control reaction yielding colorless productsare usually between 0.05 and 0.1. Then the threshold level is determinedfor that particular reaction to be 3.5 times the value of the negativecontrol. A well is considered weakly positive if the reaction yields anO.D. reading that exceeds 3.0-fold but is below 3.5-fold of the negativecontrol reading. A weakly positive well is rejected if two otherstrongly positive alleles are present for that locus. In the absence oftwo other strongly positive alleles for each locus, the weakly positivewell is accepted if it is confirmed with repeat testing or alternativemethods. A truly positive well is assigned when the O.D. readings exceed3.5-fold over the value of the negative control. The computer programanalyzes the results of all the wells, determines the positive wellsbased on the established criteria, and assigns the alleles based onwhich primer pairs exist in the positive wells. If more than two allelesare identified per locus, then the results have to be analyzed using thefollowing protocol and confirmed by repeat testing or alternativemethods.

[0107] By storing numerical reading values for the various primer pairs,many different type of assessment are possible. For example, the effectsof the changes in primer pairs and primer sequences on average O.D.readings can be assessed. Consistently weak reacting sets can bereplaced with primer pairs giving more robust and consistent results.Alternatively, if a particular weak reacting set of primers have nosubstitute, then handicap scores can be given. A more consistent traycan be developed by using the reading values as a point of reference.

[0108] VIII. High Throughput Methods and Systems.

[0109] In the present invention, high-throughput analysis of HLAgenotypes can be performed using automated devices. For example, anautomated workstation (see e.g., U.S. Pat. No. 5,139,744, “Automatedlaboratory workstation having module identification means”) can be usedto perform many of the steps involved in the present invention. An“automated workstation” is typically a computer-controlled apparatuswhich can, through robotic functions, transfer, mix, and remove liquidsfrom microtiter plates. An automated workstation can also contain abuilt-in plate reader, which can read the absorbance of a liquid in amicrotiter well. The automated workstation can be programmed to carryout a series of mixing, transfer, and/or removal steps. The automatedworkstation will typically have a multi-channel pipettor which canpipette small amounts of liquid (e.g., microliter amounts) from a vesselto the well.

[0110] For example, in some embodiments of the present invention, theautomated workstation can be used to transfer DNA samples,oligonucleotides, amplification reagents. The automated work station canalso be used to wash the samples using wash buffer. In addition,detection of oligonucleotides containing a detectable label can becarried out using an automated workstation. For example, the automatedworkstation can be used to add a detection reagent to the wells. Theautomated workstation, when equipped with a plate reader, can monitorthe absorbance of the reaction of the detection reagent in the wells.

[0111] A number of robotic fluid transfer systems/automated workstations are available, or can easily be made from existing components.For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.)automated robot using a Microlab 2200 (Hamilton; Reno, Nev.) pipettingstation can be used to transfer parallel samples to 96 well microtiterplates to set up several parallel simultaneous ligation reactions. Otherautomatic microplate dispensers include Lambda Jet and Lambda Dot (OneLambda, Inc. CA), and various other automatic plate washers anddispensers (e.g. from Thermo Labsystems, Inc. or Molecular Devices,Inc.). Moreover, it will be apparent to those of skill in the art thatthe PCR setup, reagent addition and washing steps can be automated withexisting robotics outlined above.

[0112] Optical images viewed (and, optionally, recorded) by a camera orother recording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image, e.g., using PC (Intel x86 or Pentiumchip-compatible DOS OS2 WINDOWS, WINDOWS NT or WINDOWS 98 basedmachines), MACINTOSH, or UNIX based (e.g., SUN work station) computers.

[0113] One conventional system carries light from the specimen field toa cooled charge-coupled device (CCD) camera, in common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g. individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques. The use of such automated machines, can minimizethe existence of false positives, labor requirements, variabilities,human errors, human subjectivity, and human expertise requirements, andmaximizes throughput, accuracy, sensitivity and specificity.

[0114] IX. Hybridization of Capture Oligonucleotides to HLAAmplification Products

[0115] Hybridization of Immobilized Capture Oligonucleotides to HLAAmplification Products

[0116] This method involves the use of immobilized oligonucleotides tocapture HLA allele sequences contained in an amplification product.Briefly, HLA allele sequences are amplified from a template nucleic acidusing HLA allele-specific forward and reverse primers. One or both ofthe amplification primers can contain a detectable label. Then theamplification products are denatured and hybridized to a locus-specificor allele-specific capture oligonucleotide that is already immobilizedto a solid phase to form a detectable complex. The presence of thedetectable label in the detectable complex is then measured usingmethods known to those of skill in the art (e.g., spectrophotometricmeans, a luminometer, etc.), which may require the addition of one ormore detection reagents (e.g., an avidin-enzyme molecule with acolorimetric enzyme substrate).

[0117] The capture oligonucleotides possess sufficient nucleotidecomplementarity to the HLA allele sequences being amplified that theycan hybridize to them under stringent conditions. Typically, the HLAallele-specific forward an or reverse primer will contain a detectablelabel (e.g., biotin, digoxigenin, EDANS, or a fluorescent moiety, etc.)so as to facilitate detection. Thus, this method allows for theamplification of many different HLA alleles which can be detected with,in the case of some HLA loci, as little as one capture oligonucleotidethat is locus-specific. This is an advantage over previous methods, inwhich allele-specific capture oligonucleotides were used, as thedetection of hundreds of alleles would require hundreds ofallele-specific capture oligonucleotides (see e.g., Erlich et al. (1991)Eur. J. Immunogenet. 18(1-2): 33-55; Kawasaki et al. (1993) MethodsEnzymol. 218:369-381). Thus, the present invention permits a greatsimplification and reduction in the number of oligonucleotides requiredto detect hundreds of HLA-alleles.

[0118] Hybridization of Free Capture Oligonucleotides to HLAAmplification Products and Subsequent Immobilization of the DetectableComplex

[0119] In another embodiment of the present invention, the hybridizationtakes place in solution with capture oligonucleotide(s) and then thecapture oligonucleotide is immobilized. This method involves the use ofcapture oligonucleotides that are hybridized in solution to HLA allelesequences contained in an amplification product and subsequentimmobilization of the capture oligonucleotide to a solid phase. First,HLA allele sequences are amplified from a nucleic acid using HLAallele-specific forward and reverse primers. Then the amplificationproduct are denatured and hybridized to a locus-specific orallele-specific capture oligonucleotide that is already immobilized to asolid phase. The capture oligonucleotides then hybridize and bind to thedenatured single stranded PCR products at a suitable hybridizationtemperature and “capture” complementary sequences in the products ontothe plate. If none or very little complementary sequences for thecapture oligonucleotide are present after the nucleic acid amplificationreaction (for example, if the allele sequence represented by theallele-specific PCR primers are not present in the sample DNA template,then no PCR product would be formed), then it is unlikely a detectablecomplex will form. The capture oligonucleotides possess sufficientnucleotide complementarity to the HLA allele sequences being amplifiedthat they can hybridize to them under stringent conditions. Typically,the HLA allele-specific forward and/or reverse primer will contain adetectable label (e.g., biotin, digoxigenin, EDANS, or a fluorescentmoiety, etc.) so as to facilitate detection.

[0120] In this method, capture oligonucleotides with either conservedsequences (e.g., locus-specific oligonucleotides) or allele specificsequences can be used. The later offering an additional level ofspecificity whereas the former offers convenience and ease of setup aswell as lower cost in having fewer sets of oligonucleotides. Thus, thismethod allows for the amplification of many different HLA alleles whichcan be detected with, in the case of some HLA loci, as little as onecapture oligonucleotide that is locus-specific. This is an advantageover previous methods, in which allele-specific capture oligonucleotideswere hybridized in solution to a locus-specific HLA amplificationproduct, as the detection of hundreds of alleles would require hundredsof allele-specific capture oligonucleotides (see e.g., Nevinny-Stickeland Albert (1993) Eur. J. of Immunogenet., 20: 419-427). Thus, thepresent invention permits a great simplification and reduction in thenumber of oligonucleotides required to detect hundreds of HLA-alleles.

[0121] X. Amplification of HLA Sequences With Immobilized Primers

[0122] This method involves the amplification of HLA sequences usingallele-specific primers, where one of the pair of amplification primersis immobilized to a solid phase. The other primer constituting theprimer pair contains a detectable label and is initially free insolution. This technique is not limited to the detection of HLA alleles.Essentially, any set of amplification primers and any gene can beamplified. With this method, the immobilized amplification primer servesto immobilize the amplification product directly to a solid phase. Theamplification should only take place if allele that can be amplifiedwith a particular pair of allele-specific primers is present insolution. The nucleic acid amplification and capture of PCR product takeplace on the same polycarbonate plate and the captureoligonucleotide/PCR primer is an allele specific sequence thatidentifies the sequence of interest (e.g. the particular HLA allele) andserves three purposes. First, it serves as the capture oligonucleotideand immobilizes the PCR products onto the plates. Second, it serves asone of the PCR primers that facilitate the nucleic acid amplificationreaction. Third, it serves as the discriminating sequence that allowsidentification of the correct allele. This means that the PCRamplification reaction would only take place if the correct sequencesthat is perfectly complementary to the template (which is the particularallele of the person whose HLA sequence or other sequence is beingtyped) is present on both PCR primers. An advantage of this method isthe elimination of transfer, reduction of an additional set ofoligonucleotides to the assay vessel (compared with two previous methodsdescribed under Section X).

[0123] If a sequence specific nucleic acid amplification reactionoccurred due to perfect matching between the PCR primers and thetemplate sequences, then the product would be immobilized on the solidphase. Following capture, the unbound non-specific labeled PCR primerscan be washed off With fluorescent probes, the plate can be read with anautomated fluorescent ELISA format reader. With colorimetric reactionsthat are associated for example with avidin conjugated enzyme andsubstrate systems (e.g. avidin-conjugated horseradish peroxidase andTMB), a photometric ELISA format reader would be able to quantitate theresult.

[0124] XI. Multiplexing of Positive Controls

[0125] In certain embodiments, one or more positive control can be addedto each reaction vessel. For example, a positive control in every wellcan be used to distinguish from the allele specific reactions by virtueof having a different fluorophore or enzyme-substrate combinations. Forexample, if the allele specific reaction and the positive control usedifferent fluorophores, then the excitation and emission wavelengths forboth fluorophores can be used. The positive control amplified fragmentwould be longer than the allele specific reaction so that the allelespecific reaction would be favored. The positive controls would becaptured by the same capture probe as the allele specific if the captureprobe is locus-specific. If allele-specific capture probes are used,then the positive controls may have complementary sequences to theallele specific capture probes at its 5′ end of the primer that islabeled.

[0126] XII. Magnetic Bead Variation

[0127] This method takes advantage of a commercially available nucleicacid purification method that employs magnetic beads coated with avidinor other materials to facilitate the “fishing” of the appropriatenucleic acid product of interest (KINGFISHER™ available from ThermoLabsystems, Inc.). For example, if biotinylated oligonucleotide PCRprimers are used, then a biotinylated PCR product will be captured withthe avidin on the beads. The magnetic beads are then pulled out of thereaction well, washed and all non-biotinylated materials will be washedoff. The biotinylated products and primers are then separated fromavidin coated beads by further treatment, such as elution with excessfree biotin. Thereafter, the biotinylated products are hybridized to thecapture probe of interest and separated from the biotinylated primers.Alternatively, a labeled hybridization probe is allowed to bind to thePCR product, followed by washing using the KINGFISHER™ method to removeany unbound non-specific signals. Lastly, the signals would be measured.Instead of biotinylated beads, covalently modified beads that attach toPCR oligonucleotides can also be used.

[0128] XIII. SSOP With Molecular Beacon Detection

[0129] In the methods of the present invention, molecular beaconoligonucleotides can be used to hybridize with allele-specificamplification products. Once the modular beacons are hybridized to acomplementary sequence in an amplified product, the quencher group is nolonger close enough to quench the fluorophore. As a consequence thefluorophore can be detected and quantitated. These molecular beaconoligonucleotides are known in the art and can be readily designed (seeMaterials section on design and construction of molecular beaconoligonucleotides). These oligonucleotides have the advantage of beingdirectly assayable with a device that can measure fluorescence. Inaddition, this method can exhibit lower background signal than othermethods as only oligonucleotides that are incorporated into anallele-specific product will give off a signal. Thus, molecular beacondetection does not require the addition of a detection reagent toobserve whether an HLA genotype is present in an analyte.

[0130] XIV. In Situ Amplification Variation

[0131] In certain embodiments, the in situ amplification method ischosen to eliminate the need for DNA extraction and preparation. Incontrast to the usual limitations of in situ amplification where thenumber of cycles has to be curtailed to prevent the floating away ofamplified products from the cell, it is irrelevant whether amplifiedproduct stays in the cell or out. As a result, the same number of cyclescan be used to generate the same degree of amplification as traditionalPCR. If molecular beacon method is not incorporated into the protocol,then the reaction products from the wells will be transferred to anothermicrotiter plate that has surface attached capture oligonucleotideprobes that are similar to the ones described earlier with eitherconserved sequences which can be used in all the wells or allelespecific sequences. By using an in situ amplification method it is thenpossible to use molecular beacons to detect the amplified products. Insitu amplification can be carried out on a microscopic slide, a tissuesample, a microtiter plate, etc.

[0132] The molecular beacon method can be incorporated to eliminate eventhe washing step as well as the need for specially modified plates thatcan be quite expensive. It also allows for real time measurement of PCRproduct formation. When PCR products are formed and denatured during thevarious cycling steps, molecular beacons would hybridize to some of thecomplementary single strands, thereby fluorescing and allowing real timemeasurement. If real time measurement is not desired, then the molecularbeacon probe can be added at the end of the reaction and only wells withamplified products that are complementary to the molecular beacon wouldlight up. Because the unbound molecular beacon does not fluoresce,washing steps may not be necessary if the signal to noise ratio is highenough.

[0133] XV. Tissue Block Section Variation

[0134] The methods of the present invention can be carried out onparaffin embedded formaldehyde fixed sections of buffy coats, umbilicalcord blood clots or blood clots placed onto glass slides with grids. Thesame sample can be placed onto one slide and different probes are usedin an in situ method or many samples can be placed onto the same slideand the same probe is used for all the samples. In the latter, as manysections and slides of the samples will be cut as the number of probesplus controls. This method appears to be easier for the amplification,since there is no need to separate the different probes or reactionsfrom one another.

EXAMPLES Example 1 Detection of HLA Alleles With Pre-Immobilized HLALocus-Specific Capture Oligonucleotides

[0135] As a first step, experiments were carried out to determine whatare the optimum conditions for immobilizing a capture oligonucleotide toa plate. In this experiment, Capture Oligonucleotide1(5′ACCGCACCCGCTCCGTCCCATTGAAGAAAT) was modified with an amine at the 5′end with a C6 linker and a biotin group on the 3′ end. For the purposeof actual HLA genotyping, the Capture Oligonucleotide will not have abiotinylated 3′ end. The oligonucleotide1 was incubated on a 96 wellCovalent Binding Microwell plate (Xenobind™, Xenopore, Hawthorne, N.J.)according to the manufacturer's instructions. The plate was then washedthree times with phosphate-buffered-saline (PBS). ExtrAvidin® Peroxidase(SIGMA) was added and allowed to incubate on the tray. The plate waswashed three times with PBS. TMB substrate(3,3′,5,5′-Tetramethylbenzidine) was added to the plate, 1N HCl addedand tray was read at 450 nm. The current optimum conditions foroligonucleotide binding was Capture Oligonucleotide at 100 ng/ul in PBSat pH 8.8 incubated overnight at 4° C. Alternatively, binding can occurat 37° C. for 2 hours with Capture Oligonucleotide at 100 ng/ul in PBSat pH 8.8.

[0136] Amplification of HLA alleles was carried out on DNA extractedfrom cord blood from three donors: Sample #8, Sample #12, and Sample#18. Purification of the DNA was carried out using either the Saltextraction with ethanol precipitation method or the Qiagen QIAamp®isolation method. The amplification was carried out usingoligonucleotide primers designed to hybridize to alleles in the HLA A,B, C loci for Class I and HLA DR and DQ for Class II. The sequences andlocation of these primers are given in Tables 1 & 2. For Examples 1, 2,and 3, the primers listed in Tables 5 and 6 were biotinylated.

[0137] All primers are adjusted to their optimum concentration of 100ng/ul. Primer pair mixes were set up to aliquot into PCR trays. Twodifferent 96 well trays are set up see Tables 3 & 4. The mixes arealiquoted into labeled 1.2 ml according to the volumes given in Tables 3& 4.

[0138] A 96 well tray dotting machine was utilized to dot the PCR Trays.The polypropylene trays are labeled with their tray identification,i.e., Class I tray and dotting number. 200 trays can be dotted with each1.1 ml Primer Mix set. The 96 well dotting machine was adjusted to adraw volume of 250 ul and a dispense volume of 5 ul. Fifty 96 well traysat a time can be dotted. Once the primers are dotted 17.0 ul of mineraloil was added to each well. The PCR tray was then covered with adhesivetape. The trays are then boxed and stored at −20° C. until use.

[0139] HLA allele sequence amplification was accomplished by adding theDNA mixture to the PCR tray and placing the tray in a thermal cyclingoven. The DNA mixture contains: 40.0 ul of DNA (50-100 ng/ul), 4.0 ulTaq polymerase (5 U/ul), and 600.0 ul PCR Mix into a labeled 1.5 mltube. For Class I HLA trays, the PCR mix contains 30 mM AmmoniumChloride, 150 mM Tris-HCl pH 8.8, 4 mM MgCl₂, and 166 uM dNTP. For ClassII HLA trays, the PCR mix contains 100 mM KCl, 20 mM Tris HCl pH 8.8,0.2% Triton X-100, 3.4 mM MgCl₂, and 166 uM dNTP.

[0140] A liquid sample dispensing machine was used to add the DNAmixture to tray PCR tray. The 250 ul dispensing syringe was employed.The machine was set to add 5.0 ul to a 96 well microtiter tray. Theappropriate PCR tray was placed in the machine. The DNA mixture wasvortexed and then 5.0 ul of DNA mixture was dispensed into each of the96 wells of the PCR tray. The tray was then placed in the thermalcycling oven (BioOVen, BioTherm™ Products, MD). The PCR was carried outin the cycling oven in the following 6 stage program: 1.)  1 Cycle 97°C. for 20 seconds 2.)  5 Cycles 97° C. for 35 seconds, 61° C. for 45seconds, 72° C. for 40 seconds 3.) 25 Cycles 97° C. for 20 seconds, 59°C. for 45 seconds, 72° C. for 40 seconds 4.)  4 Cycles 97° C. for 20seconds, 57° C. for 45 seconds, 72° C. for 90 seconds 5.)  1 Cycle 72°C. for 4 minutes 6.)  1 Cycle 30° C. for 1 second

[0141] This 6-stage program generates the optimum PCR amplificationprofile for this example. After amplification, PCR product was diluted.A dilution of 1:10 with PBS pH 7.4 was optimum. Therefore, 90 ul of PBSpH 7.4 was added to the PCR product. 50.0 ul of diluted PCR product wastransferred from the PCR tray to the Capture plate using the 96 welldotting machine. The machine was adjusted to draw and dispense 50.0 ul.

[0142] The capture tray was then placed in the thermal cycling oven andthe one stage Capture Program was run. The Capture program for thisexample was as follows:. 1 Cycle of 97° C. for 6 minutes, 57° C. for 12minutes, and 30° C. for 1 second. 100 ul of hybridization solution (PBSat pH 7.4) was added to the capture tray. Also a hybridization solutionof 0.9 M NaCl, 90 mM sodium citrate, 1 mM EDTA, 0.1% Ficoll, 0.3% BSA,0.5% SDS can be used. The tray was incubated at 45° C. for 120 minutes.After the hybridization incubation the capture plate was washed. Usingthe plate washer, the capture plate was rinsed three times with 200 ulPBS pH 7.4 in each well.

[0143] For detection, ExtrAvidin® Peroxidase was diluted 1:2000 in 4%BSA in PBS pH 7.4, and 50.0 ul was added to each well. The Capture traywas incubated at 37° C. for 30 minutes. Then the Capture tray was washedfour times with 200 ul PBS pH 7.4 in each well by the plate washer. 50.0ul of liquid substrate (3,3′,5,5′-Tetramethylbenzidine) was added toeach well and incubated at 37° C. for 30 minutes. 50.0 ul of 1N HCl wasadded to each well to stop the reaction. The trays are read on the Platereader by setting the filter to 450 nm. The plate configuration was setto default a 96 well Flat bottom microtiter plate.

[0144] Data readings are stored as a spreadsheet file. Positivereactions are identified by values over threshold. Threshold wasdetermined by numerical values that are at least 3.5 times over thevalue of the negative control and the average of the negative reactionvalues. HLA typing results are determined by the specificitycorresponding to the positive reactions. The genotypes were determinedas follows:

[0145] Sample #8 A*0201,A*2402, B*0701, B*3501 C*0401,DRB1*0101,DRB1*1501

[0146] DRB5*0101: Sample #12, A*0201, B*1301, B*4402, C*0601 DRB1*0403,

[0147] DRB1*1401 DRB3*0101,DRB4*0101; and Sample #18 A*0101,A*1101

[0148] B*0801,B*1801, C*0701, DRB1*0901, DRB1*1403, DRB3*0301,DRB4*0101.

Example 2 Simultaneous Hybridization of Capture Oligonucleotide toDenatured PCR Product to Capture Plate.

[0149] For this example, a modification of the method carried out inExample 1 was performed. In this example, the amplification product ishybridized to a capture oligonucleotide(s) in solution. The captureoligonucleotide is then immobilized on a solid phase. The complexes arewashed and a detection step is then performed.

[0150] The set-up of the PCR Tray was carried out as in Example 1. ThePCR amplification was carried out as in Example 1 on DNA from donors #8,#12, and #18. The DNA was purified as in Example 1. After PCRamplification, diluted capture oligonucleotide was added to the wells:5.0 ul of capture oligonucleotide at a concentration of 50 ng/ul wasadded to each well. The tray was placed in a thermal cycling oven andsubjected to the following capture program: 1 Cycle of 97° C. for 20seconds, 57° C. for 60 seconds, and 30° C. for 1 second. After thecapture program is run, the PCR products are now hybridized with thecapture oligonucleotide. The hybridized PCR products are diluted. Adilution of 1:10 with PBS at pH 7.4 was optimum. 90.0 ul of PBS at pH7.4 was added to each well in the PCR tray. 15.0 ul of the diluted PCRproduct was transferred by the 96 well dotting machine into a newcovalent binding plate (Xenobind™) containing 50.0 ul of PBS at pH 7.4in each well. The plate was incubated overnight at room temperature sothat the hybridized PCR product with the capture oligonucleotide withits amine linker at the 5′ end can bind to the plate.

[0151] Using the plate washer, the plate was washed twice with 0.1% BSAin PBS at pH 7.4. ExtrAvidin® Peroxidase conjugate was diluted 1:2000 in4% BSA in PBS at pH 7.4, and 50.0 ul was added to each well. The platewas incubated at 37° C. for 30 minutes and then washed six times with200 ul of PBS pH 7.4 in each well by the plate washer. 50.0 ul of liquidsubstrate (3,3′,5,5′-Tetramethylbenzidine) was added to each well andincubated at 37° C. for 30 minutes. 50.0 ul of 1N HCl was added to eachwell to stop the reaction. The Tray reading was carried out as inExample 1. The Analysis is carried out as in Example 1.

[0152] Four basic results were observed. A “Good” result was assigned ifthe value for the negative control was the same as the value of anegative allele specific primer pair. Also the value of the positivecontrol had to be higher than the value of the negative control by afactor of at least 3.5. Furthermore, the value of all positive wells hadto be 3.5 times greater than the negative wells. A “Weak” result wasassigned if the signal to noise ratio is above three fold but less thanthe 3.5 fold necessary for comfortable discrimination between positivereactions and negative reactions. Results were identified as “TooPositive” or “Background” if the value of the negative control waswithin acceptable limits but some of the negative wells have valuesequal or above that of the positive control wells. Results of “TooPositive” were observed when the Avidin conjugate concentration was toohigh or if insufficient washing was performed or if there was PCR DNAcontamination. An “All Negative” result would be assigned if the valuesof the all wells were similar to the value of the negative control well.Results of “All negative” were observed when hybridization temperatureswere too stringent (above 45° C.) or if the hybridization incubationtimes were too short (less than one hour) or if the washing conditionswere too vigorous. Dilution and washing conditions are important factorsto obtain the best conditions. If the hybridization product was notdiluted enough, non-specific binding would result in false positives. Ifthe washes were not exhaustive enough, false positive results would beobserved.

[0153] The use of the automatic plate washer eliminated the inconsistentresults and false positives that results from accidental PCR productcontamination that manual handling produces. Once the washer wasemployed, false positive reactions and false negative reactions weregreatly reduced. This observation is most likely and logicallyattributed to the elimination of carryover and inconsistent washing thatoccurs with manual washing.

[0154] In parallel with the procedure just carried out, PCR-SSP wasperformed using the same primer pair sets and amplification conditions.Briefly, PCR-SSP was performed with the primers sets described and theamplification products were run on agarose gels. The bands on the gelidentified the positive reactions and a typing was obtained based on thepositive reactions.

[0155] The allele assignments of donors #8, #12, and #18 using thePCR-SSP method and the inventive method of this example are given below:

Summary of Typing Results

[0156] Sample #8:

[0157] PCR-SSP: A*0201,A*24XX B*07XX, B*3501 C*0401 DRB1*0101 DRB1*1501DRB5*0101.

[0158] Inventive Method: A*0201,A*2402, B*0701, B*3501, C*0401,DRB1*0101, DRB1*1501, DRB5*0101.

[0159] Sample #12:

[0160] PCR-SSP: A*0201, B*1301, B*44XX, C*0601, DRB1*0403, DRB1*1401,DRB3*0101, DRB4*01XX.

[0161] Inventive Method: A*0201, B*1301, B*4402, C*0601, DRB1*0403,DRB1*1401, DRB3*0101, DRB4*0101.

[0162] Sample #18:

[0163] PCR-SSP: A*0101,A*1101, B*0801, B*1801, C*0701,DRB1*0901,DRB1*14XX, DRB3*03XX, DRB4*01XX.

[0164] Inventive method: A*0101, A*101, B*0801, B*1801, C*0701,DRB1*0901, DRB1*1403, DRB3*0301, DRB4*0101.

[0165] The HLA typing from the two methods matched and was found to bein total correlation. With these samples there was 100% specificity,that is, all positive controls or expected positive samples weredetected as positive reactions with readings that were at least 3.5 foldthat of negative values, and all expected negative controls or samplesproduce negative results. 100% sensitivity was also observed with theappropriate positive readings for the positive controls or expectedpositive samples.

[0166] The HLA nomenclature at the allelic level is as follows. Thefirst letter denotes the locus, i.e. HLA A and B for Class I, or DRB forClass II. The asterisk (*) denotes DNA typing. The first two numbersdesignates serological level or equivalent assignments. The third andfourth numbers are the allele level subtypes that are distinguished byDNA typings. The fifth and sixth numbers are usually not displayedbecause these designate silent mutations, i.e. DNA substitutions that donot produce changes in protein sequence coding of the final HLA proteinantigen. The seventh number, which is usually not displayed as well,denotes a null mutation, which is a mutation that silences theexpression of the allele at the protein or mRNA level. There are one totwo potential alleles at each locus; however, in homozygous situationswhere both alleles are identical, only one allele can be identified andtyped. Where there is an XX after the first two numbers, it means thatonly one allele can be identified. This usually means that there may behomozygosity, but in a small number of cases, there may mean that thereis a allele that was not detected by the entire panel of primers eitherbecause the panel cannot be all inclusive or because the allele is newand previously undiscovered.

[0167] In all instances, positive reactions observed on the PCR-SSPagarose gels corresponded to positive OD values that are at least3.5-fold that of negative controls or negative wells on the platereader. In this respect, it is instructive to note that because theinventive method of this example is amenable to larger sets of primerpairs, it detects several of the alleles at a higher level of resolutionthan the PCR-SSP method. Hence, there were several XX assignments forthe third and fourth numbers in some of the alleles tested by PCR-SSP.However, the PCR-SSP method is fully capable of typing every sample tothe same degree of resolution as the inventive method of this exampleeven though is far more laborious.

Example 3 Amplification of HLA Sequences With an ImmobilizedAllele-Specific Primer.

[0168] This method involves the amplification of HLA sequences usingallele-specific primers, where one of the pair of amplification primersis immobilized to a solid phase. The other primer constituting a primerpair contains a detectable label and is initially free in solution.Reference DNAs were used as the template nucleic acid. The referenceDNAs are from a panel of DNA that was used for the UCLA DNA ExchangeProgram. Primers directed to detecting class II HLA alleles were used inthis example. In this example, the following immobilization primerscontained an amine group followed by a C6 linker: SEQ ID NO: 189, DR06,CGTTTCTTGGAGCAGGCTAAGTG; SEQ ID NO: 190, DR07, CGTTTCTTGGAGTACTCTACGGG;SEQ ID NO: 191, DR08, ACGTTTCTTGGAGCAGGTTAAAC; SEQ ID NO: 192, DR09,CGTTTCCTGTGGCAGCCTAAGA; SEQ ID NO: 193, DR10, CGTTTCTTGGAGTACTCTACGTC;and SEQ ID NO: 277, DRCPT1, TGGCGTGGGCGAGGCAGGGTAACTTCTTTA. The primerswere immobilized to a Xenobind™ (Covalent Binding Microwell Plates),Xenopore, Hawthorne, N.J.) plate according to the manufacturer'sinstructions. The DNA samples were isolated from reference samples knownHLA allele sequences. The amplification buffer and components are thesame as in Example 1 for the class II amplification. The buffercontaining Taq and the proper amplification reagents were added to themicrotiter wells. The other member of the primer pairs were biotinylatedat their 5′ ends and were as follows: SEQ ID NO:222, DR39,TGCACTGTGAAGCTCTCAC, SEQ ID NO:223, DR40, CTGCACTGTGAAGCTCTCCA. Theprimers were paired in separate microtiter wells as follows for sample219 and sample 223: Mix Primer 1 Primer 2 Specificity 1 DR09 DR39 DR16 2DR09 DR40 DR15 3 DR10 DR39 DR 3A, 11A, 13A, 14A 4 DR10 DR40 DR 3B, 11B,13B, 14B 5 DR08 DR39 DR 4A 6 DR08 DR40 DR 4B 7 DR07 DR39 DR 8 8 DR07DR40 DR12 9 DR06 DR39 DR53 10  Drcapt1 DR39, 40 positive control 11 none none none 12  none none none

[0169] The amplification program was carried out as in Example 1. Afteramplification, the plate was washed twice with 0.1% BSA in PBS at pH7.4. ExtrAvidin® Peroxidase conjugate was diluted 1:2000 in 4% BSA inPBS at pH 7.4. 50.0 ul was added to each well. The plate is incubated at37° C. for 30 minutes and then washed six times with 200 ul of PBS pH7.4 in each well by the plate washer. 50.0 ul of liquid substrate(3,3′,5,5′-Tetramethylbenzidine) is added to each well and incubated at37° C. for 30 minutes. 50.0 ul of 1N HCl is added to each well to stopthe reaction. In parallel with the immobilized PCR method justdescribed, PCR-SSP using the above listed primers pairs was carried outand the samples were typed by running them on agarose gels. The resultsof the PCR-SSP typing method and the immobilized PCR primer methodcarried out in this example were in complete agreement. The expectedtyping of the reference DNA and the genotypes determined using PCR-SSPand immobilized PCR of this example were the same: HLA Genotype ofGenotype determined by PCR-SSP DNA ID the Reference DNA and theInventive Method 219 DR1501, DR0404 DR15, DR04B 223 DR1101, DR0403 DR3,11, 13, 14A, DR04B

[0170] Thus, this example shows that PCR can be carried out with animmobilized primer to successfully genotype samples for their HLA allelesequences.

Example 4 Multiplexing of Positive Controls Into Every Well

[0171] A positive control in every well can be used to distinguish fromthe allele specific reactions by virtue of having a differentfluorophore or enzyme-substrate. For example, if the allele specificreaction and the positive control use different fluorophores, then theexcitation and emission wavelengths for both fluorophores will be used.The positive control amplified fragment will be longer than the allelespecific reaction so that the allele specific reaction would be favored.The positive controls would be captured by the same capture probe as theallele specific if the capture probe is conserved. If the allelespecific capture probes are used, then the positive controls may havecomplementary sequences to the allele specific capture probes at its 5′end of the primer that is labeled.

[0172] In this method, positive control primers would be used. Forexample, SEQ ID NO:270:5′DPA-E (PC), 5′GATCCCCCTGAGGTGACCGTG and SEQ IDNO:271: 3′DPA-F (PC), 5′CTGGGCCCGGGGGTCATGGCC are used. SEQ ID NO: 270would be labeled with the amine linker at the 5′ end and is designated5′PC. SEQ ID NO: 271 is the 3′ positive control primer and would belabeled with a fluorophore (e.g., fluorescein at the 5′ end) and isdesignated 3′PC-(CTGGGCCCGGGGGTCATGGCC). These primers can be added toPCR mixes and used as internal controls in each well by detected theirspecific fluorescent signal.

Example 5 Detection of HLA Sequences Using Molecular Beacon Probes

[0173] Molecular beacon probes could be used to detect allele-specificamplification products. Briefly, amplification of HLA allele sequencesusing HLA-specific primers is first carried out. Then molecular beaconprobes that hybridize with HLA alleles sequences are hybridized todenatured amplification products. If the molecular beacon probehybridizes then the fluorophore is no longer quenched and fluorescencewould be exhibited and detected.

[0174] Fluorophore-quencher probes would be constructed from the HLAsequences given in Table 1 and 2. The loop portion of the probe would beconstructed so that the sequence matched the polymorphic sequences ofthe HLA sequences similar to the sequences given in Tables 1 & 2. At the5′ termini there a would be 5 nucleotides of T ending with thefluorophore (e.g. 5-(2′-aminoethyl) aminonapthtalene-1-sulfonic acid(EDANS) at the 5′ end. At the 3′ end there would be a poly-A tail of 5nucleotides ending with the quencher (e.g.4-(4′-dimethylaminophenylazo)-benzoic acid (DABCYL) at the 3′ end.

[0175] Following PCR amplification, the products are denatured byincubating them at 100° C. for 10 minutes and then diluted inhybridization buffer. Diluted Class I products are added to theMolecular Beacon tray containing the Class I fluorophore and quencherprobes. Similarly, the Class II diluted PCR product is added to theClass II Molecular Beacon tray.

[0176] To make up the tray containing the molecular beacon primers,0.5-1.0 uM concentration of molecular beacon primers are made. Themolecular beam primers would be added to wells containingallele-specific amplification products. The Molecular Beacon tray isallowed to incubate at 45-57° C. for a period of time to allow forhybridization.

[0177] When the complementary target is encountered the fluorophore isexposed and the probe can fluoresce. The tray is read by a fluorescentreader with the excitation set at 336 nm and the emission set at 490 nm.Positive reactions are identified by strong fluorescent reading and datareadings are stored as a spreadsheet file. Positive reactions areidentified by values over threshold. Threshold is determined bynumerical values that are at least 3 times over the value of thenegative control and the average of the negative reaction values.

Example 6 In Situ Amplification Variation

[0178] Oligonucleotide primers will be used that are designed tohybridized to the polymorphic regions of HLA A, B, C loci for Class Iand HLA DR and DQ for Class II. The sequences and location of theseprimers are given in Tables 1 & 2. The primers listed in Tables 5 and 6are biotinylated. All primers are adjusted to their optimumconcentration of 100 ng/ul. Primer pair mixes will be set up to aliquotinto PCR trays. Two different 96 well trays will be set up see Tables 3& 4. The mixes will be aliquoted into labeled 1.2 ml tubes according tothe volumes given in Tables 3 & 4. A 96 well tray dotting machine isutilized to dot the PCR Trays. The polypropylene trays are labeled withtheir tray identification, i.e., Class I tray and dotting number. 200trays can be dotted with each 1.1 ml Primer Mix set. The 96 well dottingmachine is adjusted to a draw volume of 250 ul and a dispense volume of5.0 ul. Fifty 96 well trays at a time can be dotted. Once the primersare dotted 17.0 ul of mineral oil is added to each well. The PCR tray isthen covered with adhesive tape. The trays are then boxed and stored at−20° C. until use.

[0179] The sample would be a cell prep containing nucleated cells, or acrude cell prep with inhibitory proteins (heme) removed. First, 50-100mg of cell prep are diluted in 100-200 ul of dH20. Then Proteinase K (20mg/ml) (Fisher Scientific) would be added (100 ul is used for every 50mg of cell prep) and the sample is incubated to digest proteins in thesample. The lysate sample is incubated at 100° C. for 1 minute toinactivate the Proteinase K.

[0180] PCR amplification would be accomplished by adding the DNA mixtureto the PCR tray and placing the tray in a thermal cycling oven. For DNAmixture aliquot-lysate sample, 4.0 ul Taq polymerase (5U/ul), and 600.0ul PCR Mix into a labeled 1.5 ml tube and place on ice. The PCR buffersare the sample as in Example 1: For Class I trays, PCR Mix—30 mMAmmonium Chloride, 150 mM TRIS-HCl pH 8.8,4 mM MgCl₂, and 166 uM dNTP;For Class II trays, PCR Mix—100 mM KCl, 20 mM TRIS HCl pH 8.8, 0.2%Triton X-100, 3.4 MM MgCl₂, and 166 uM dNTP.

[0181] A Liquid Sample Dispensing machine would be used to add the DNAmixture to tray PCR tray. The 250 ul dispensing syringe would beemployed. The machine would be set to add 5.0 ul to a 96 well microtitertray. The appropriate PCR tray would be placed in the machine. The DNAmixture would be vortexed and then 5.0 ul of DNA mixture would bedispensed into each of the 96 wells of the PCR tray. The tray would thenbe placed in the thermal cycling oven.

[0182] After PCR amplification, diluted capture oligonucleotide would beadded to the wells. 5.0 ul of capture oligonucleotide at a concentrationof 50 ng/ul would be added to each well. The tray would be placed in thethermal cycling oven and a capture thermal cycle program run. After thecapture thermal cycling, the PCR products are now hybridized with thecapture oligonucleotide. The hybridized PCR products are diluted. Adilution of 1:10 with PBS at pH 7.4 is optimum. 90.0 ul of PBS at pH 7.4is added to each well in the PCR tray. 15.0 ul of the diluted PCRproduct is transferred by the 96 well dotting machine into a newcovalent binding plate containing 50.0 ul of PBS at pH 7.4 in each well.The plate would be incubated overnight at room temperature so that thehybridized PCR product with the capture oligonucleotide with its aminelinker at the 5′ end can bind to the plate. The unbound products areremoved by washing. Using the plate washer, the plate is washed twicewith 0.1% Tween 20 in PBS at pH 7.4. Avidin peroxidase conjugate isdiluted 1:2000 in 4% BSA in PBS at pH 7.4. 50.0 ul is added to eachwell. The plate is incubated at 37° C. for 30 minutes.

[0183] The plate is washed six times with 200 ul of PBS pH 7.4 in eachwell by the plate washer. 50.0 ul of liquid substrate(3,3′,5,5′-Tetramethylbenzidine) is added to each well and incubated at37° C. for 30 minutes. 50.0 ul of 1N HCl is added to each well to stopthe reaction. Trays are read on a microtiter plate reader by setting thefilter to 450 nm. The data readings would be stored as a spreadsheetfile and analyzed. Positive reactions are identified by values overthreshold. Threshold is determined by numerical values that are at least3.5 times over the value of the negative control and the average of thenegative reaction values.

Example 7 In Situ Amplification-Molecular Beacon Variation

[0184] HLA-specific Molecular beacon probes would be constructed as inExample 5. A tray of molecular beacon probes would be spotted intomicrotiter plates. The template nucleic acid is contained in a cell prepcontaining nucleated cells or a crude cell prep with inhibitory proteins(heme) removed. First, 50-100 mg of cell prep are diluted in 100-200 ulof dH20. Then Proteinase K (20 mg/ml) (Fisher Scientific) would be added(100 ul is used for every 50 mg of cell prep) and the sample isincubated to digest proteins in the sample. The lysate sample isincubated at 100° C. for 1 minute to inactivate the Proteinase K.

[0185] PCR amplification would be accomplished by adding the DNA mixtureto the PCR tray and placing the tray in a thermal cycling oven. HLAlocus-specific primers are utilized to amplify HLA Class I and Class IIproducts. For Class I primers are selected to amplify Class I exon 2 andexon 3 products. For Class II, primers are selected to amplify Class IIexon 2 products. For DNA mixture aliquot-lysate sample, 4.0 ul Taqpolymerase (5 U/ul), and 600.0 ul PCR Mix into a labeled 1.5 ml tube andplace on ice. The PCR buffers are the sample as in Example 1. FollowingPCR amplification, the PCR product is denatured by incubation at 100° C.for 10 minutes and then diluted in hybridization buffer.

[0186] Diluted Class I products would be added to the Molecular Beacontray containing the Class I fluorophore and quencher probes. Similarly,the Class II diluted PCR product would be added to the Class IIMolecular Beacon tray. The Molecular Beacon tray is allowed to incubateat 45-57° C. for a period of time to allow for hybridization. When thecomplementary target is encountered the fluorophore is exposed and theprobe can fluoresce. The tray is read by a fluorescent reader with theexcitation set at 336 nm and the emission set at 490 nm. Positivereactions are identified by strong fluorescent reading and data readingsare stored as a spreadsheet file. Positive reactions are identified byvalues over threshold. Threshold is determined by numerical values thatare at least 3.5 times over the value of the negative control and theaverage of the negative reaction values.

Example 8 Tissue Block Section Variation

[0187] The tissue block section method is a variation of the molecularbeacon method with the use of a paraffin embedded tissue sample. Theconstruction of the fluorophore-quencher probe is carried out as inExample 8 (Construction of the fluorophore-quencher probe). Molecularbeacon tray set up would be carried out as in Example 8.

[0188] The amplification of sequences on a paraffin block sample wouldoccur on a glass slide which will necessitate dotting the PCR mixes on aglass slide. Samples embedded in paraffin are sectioned and each slidewould be added to a glass slide. The specific primer mix and DNA mixturewould be added to an individual glass slide. HLA locus primers areutilized to amplify HLA Class I and Class II products. For Class Iprimers are selected to amplify Class I exon 2 and exon 3 products. ForClass II, primers are selected to amplify Class II exon 2 products.There will be 96 individual slide made to complete the Class I or ClassII sets. After adding the mix, the glass slide would be sealed with acover slip. The slides are placed in the thermal cycling oven and thePCR program for slides would be run. 1 cycle of 96° C. for 30 secondsfollowed by 34 cycles of 96° C. for 30 seconds, 61° C. for 60 seconds,72° C. for 60 seconds.

[0189] Following PCR amplification, the PCR product would be denaturedby incubation at 100° C. for 10 minutes and then diluted inhybridization buffer (0.9 M NaCl, 90 mM sodium citrate, 1 mM EDTA, 0.1%Ficoll, 0.3% BSA, and 0.5% SDS). Diluted Class I products are added tothe Molecular Beacon tray containing the Class I fluorophore andquencher probes. Similarly, the Class II diluted PCR product would beadded to the Class II Molecular Beacon tray. The Molecular Beacon traywould be allowed to incubate at 45-57° C. for 1 hour to allow forhybridization.

[0190] When the complementary target is encountered the fluorophore isexposed and the probe can fluoresce. The tray would be read by afluorescent reader with the excitation set at 336 nm and the emissionset at 490 nm. Positive reactions are identified by a strong fluorescentreading; positive reactions are identified by values over threshold.Threshold is determined by numerical values that are at least 3 timesover the value of the negative control and the average of the negativereaction values.

[0191] The data readings are then stored as a spreadsheet file. In thismanner, HLA genotyping could be achieved.

[0192] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification in their entirety for all purposes. Although the inventionhas been described with reference to preferred embodiments and examplesthereof, the scope of the present invention is not limited only to thosedescribed embodiments. As will be apparent to persons skilled in theart, modifications and adaptations to the above-described invention canbe made without departing from the spirit and scope of the invention,which is defined and circumscribed by the appended claims. TABLE 1SEQUENCE (5′-3′) Primer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18CI01 5′ HLA-C ex2 221-239 C C G A G T G A A C C T G C G G A A CI02 5′HLA-C Ex2 249-268 T A C T A C A A C C A G A G C G A G CI03 5′ HLA B & CEx2 210-228 C A C A G A C T G A C C G A G T G A CI04 5′ HLA-C Ex2123-140 A G T C C A A G A G G G G A G C C G CI05 5′ HLA-A & C Ex2 5-25 CC A C T C C A T G A G G T A T T T CI06 3′ HLA-C Ex3 243-263 T C T T C TC C A G A A G G C A C C CI07 3′ HLA-C Ex3 243-263 C A G G T C A G T G TG A T C T C C CI08 3′ HLA-B & C Ex3 195-213 C C T C C A G G T A G G C TC T C C CI09 3′ HLA-C Ex4 234-251 C A G C C C C T C G T G C T G C A TCI10 3′ HLA-C Ex3 258-275 C G C G C G C T G C A G C G T C T T CI11 3′HLA-C Ex3 195-213 C C T C C A G G T A G G C T C T C A CI12 3′ HLA-C Ex431-49 C T C A G G G T G A G G G G C T C T CI13 3′ HLA-C Ex3 134-151 T GA G C C G C C G T T T C C G C A CI14 3′ HLA-B & C Ex3 18-36 G G T C G CA G C C A T A C A T C C CI15 5′ HLA-B & C Ex3 59-76 C C G C G G G T A TG A C C A G T C CI16 3′ HLA-C Ex4 4-23 G C G T C T C C T T C C C G T T CT CI17 3′ HLA-C Ex4 4-23 A G C G T C T C C T T C C C A T T C CI18 5′HLA-C Ex3 134-151 T C C G C G G G T A T G A C C A G T CI19 3′ HLA-C Ex325-42 G C C C C A G G T C G C A G C C A A CI20 5′ HLA-C Ex2 195-213 A CA A G C G C C A G G C A C A G G CI21 3′ HLA-ABC Ex3 216-233 G A G C C AC T C C A C G C A C T C CI22 3′ HLA-A & C Ex 3 196-214 C C C T C C A G GT A G G C T C T C CI23 3′ HLA-B & C Ex3 65-84 T C G T A G G C T A A C TG G T C A CI24 3′ HLA-C Ex3 131-148 C C G C C G T G T C C G C G G C ACI25 5′ HLA-C Ex2 252-270 T A C A A C C A G A G C G A G G C C CI26 5′HLA-C Ex2 253-270 A C A A C C A G A G C G A G G C C G CI27 5′ HLA-C Ex285-103 A C G A C A C G C A G T T C G T G C CI28 3′ HLA-C Ex2 229-246 G CG C A G G T T C C G C A G G C CI29 3′ HLA-A Ex3 216-233 G A G C C A C TC C A C G C A C C G CI30 3′ HLA-ABC Ex3 216-233 G A G C C A C T C C A CG C A C G T CI31 3′ HLA-A Ex3 195-213 C C T C C A G G T A G G C T C T CT CI32 3′ HLA-A Ex3 48-64 C C G C G G A G G A A G C G C C A CI33 5′HLA-A Ex2 5-25 C C A C T C C A T G A G G T A T T T CI34 5′ HLA-A Ex2168-186 C C G G A G T A T T G G G A C C T G CI35 3′ HLA-C Ex3 25-41 C CC C A G G T C G C A G C C A G CI36 3′ HLA-B & C Ex3 169-185 C G C A C GG G C C G C C T C C A CI37 5′ HLA-B Ex2 144-161 G C G C C G T G G A T AG A G C A A CI38 5′ HLA-B Ex2 117-133 G C C G C G A G T C C G A G G A CCI39 5′ HLA-B Ex2 181-199 A C C G G A A C A C A C A G A T C T CI40 5′HLA-B Ex2 181-199 A C C G G G A G A C A C A G A T C T CI41 5′ HLA-A & BEx2 170-188 G G A G T A T T G G G A C C G G A A CI42 5′ HLA-B Ex2195-212 A A C A T G A A G G C C T C C G C G CI43 5′ HLA-B Ex2 180-199 GA C C G G A A C A C A C A G A T C CI44 3′ HLA-B Ex2 219-236 T A C C G AG A G A A C C T G C G C CI45 5′ HLA-B Ex2 157-173 A G C A G G A G G G GC C G G A A CI46 5′ HLA-B Ex2 51-68 G G G G A G C C C C G C T T C A T TCI47 5′ HLA-B Ex2 192-210 C A G A T C T A C A A G G C C C A G CI48 5′HLA-B Ex2 5-30 C C A T G A G G T A T T T C T A C A CI49 5′ HLA-B Ex2180-199 G A C C G G A A C A C A C A G A T C CI50 5′ HLA-B & C Ex2221-238 C C G A G A G A G C C T G C G G A A CI51 5′ HLA-A & B Ex2220-238 A C C G A G A G A A C C T G C G G A CI52 5′ HLA-B Ex2 116-133 CG C C G C G A G T C C G A G A G A CI53 5′ Control Primer PIC1 A T G A TG T T G A C C T T T C C A CI54 3′ Control Primer PICA T T C T G T A A CT T T T C A T C A CI55 3′ HLA-B Ex3 195-213 C C T C C A G G T A G G C TC T G T CI56 3′ HLA-B & C Ex3 44-59 G A G G A G G C G C C C G T C G CI573′ HLA-ABC Ex3 76-92 C T T G C C G T C G T A G G C G G CI58 3′ HLA-B & CEx3 77-95 A T C C T T G C C G T C G T A G G C CI59 3′ HLA-B Ex3 92-111 CG T T C A G G G C G A T G T A A T CI60 3′ HLA-B Ex3 201-218 C G T G C CC T C C A G G T A G G T CI61 3′ HLA-ABC Ex3 216-233 G A G C C A C T C CA C G C A C T C CI62 3′ HLA-B Ex3 229-246 C C A G G T A T C T G C G G AG C G CI63 3′ HLA-B Ex3 260-276 C C G C G C G C T C C A G C G T G CI643′ HLA-B Ex3 262-279 T A C C A G C G C G C T C C A G C T CI65 3′ HLA-B &C Ex3 10-29 G C C A T A C A T C C T C T G G A T CI66 3′ HLA-B Ex3 18-36C G T C G C A G C C A T A C A T C A CI67 3′ HLA-B Ex3 184-201 C T C T CA G C T G C T C C G C C T CI68 3′ HLA-B & C Ex3 69-87 G T C G T A G G CG G A C T G G T C CI69 3′ HLA-A & B Ex3 68-85 T C G T A G G C G T C C TG G T G G CI70 3′ HLA-B Ex3 156-173 C T C C A A C T T G C G C T G G G ACI71 3′ HLA-B Ex2 173-192 G T G T G T T C C G G T C C C A A T CI72 3′HLA-A & B Ex2 246-264 C G C T C T G G T T G T A G T A G C CI73 3′ HLA-BEx4 168-187 G C C C A C T T C T G G A A G G T T CI74 3′ HLA-B Ex3 11-28C C A T A C A T C G T C T G C C A A CI75 3′ HLA-B Ex2 229-245 G C G C AG G T T C C G C A G G C CI76 3′ HLA-ABC Ex3 216-233 G A G C C A C T C CA C G C A C A G CI77 5′ HLA-A Ex3 63-80 G G G T A C C A G C A G G A C GC T CI78 5′ HLA-B & C Ex2 187-205 G A G A C A C A G A A G T A C A A GCI79 3′ HLA-B Ex3 120-136 G C C G C G G T C C A G G A G C T CI80 5′HLA-B Ex2 222-239 C G A G A G A G C C T G C G G A A C CI81 5′ HLA-B Ex2119-136 C G C G A G T C C G A G G A T G G C CI82 3′ HLA-A & B Ex3228-245 C A G G T A T C T G C G G A G C C C CI83 5′ HLA-B Ex2 5-24 C C AC T C C A T G A G G T A T T T CI84 3′ HLA-B Ex3 120-136 G C G G C G G TC C A G G A G C G CI85 3′ HLA-A & B Ex3 195-213 C C T C C A G G T A G GC T C T C A CI86 3′ HLA-B Ex2 226-243 G C A G G T T C C G C A G G C T CT CI87 5′ HLA-B Ex2 244-227 G G A C C T G C G G A C C C T G C T CI88 5′HLA-B & C Ex2 52-69 G G G A G C C C C G C T T C A T C T CI89 5′ HLA-BEx2 116-133 C G C C A C G A G T C C G A G G A A CI90 3′ HLA-ABC Ex3156-172 T C C C A C T T G C G C T G G G T CI91 3′ HLA-B Ex3 44-60 G G AG G A A G C G C C C G T C G CI92 5′ HLA-B Ex2 227-244 G A G C C T G C GG A C C C T G C T CI93 5′ HLA-B Ex2 222-239 C G A G T G G G C C T G C GG A A C CI94 5′ HLA-B Ex2 76-94 G C T A C G T G G A C G A C A C G C CI953′ HLA-B Ex2 207-225 C T C G G T C A C T C T G T G C C T CI96 3′ HLA-BEx2 207-226 T C T C G G T A A G T C T G T G C C CI97 5′ HLA-A Ex2174-192 T A T T G G G A C G A G G A G A C A CI98 3′ HLA-B & C EX3 69-87C G T C G T A G G C G T A C T G G T CI99 5′ HLA-A Ex2 113-130 C G A C GC C G C G A G C C A G A A CI100 3′ HLA-ABC Ex3 216-233 G A G C C C G T CC A C G C A C T C CI101 5′ HLA-A Ex2 210-229 T C A C A G A C T G A C C GA G C G CI102 5′ HLA-A Ex2 191-209 A C G G A A T G T G A A G G C C C ACI103 5′ HLA-A Ex2 111-127 A G C G A C G C C G C G A G C C A CI104 5′HLA-A Ex2 166-184 G G C C G G A G T A T T G G G A C G CI105 5′ HLA-A Ex2152-170 G A T A G A G C A G G A G A G G C C CI106 5′ HLA-A & B Ex2210-229 T C A C A G A C T G A C C G A G A G CI107 5′ HLA-A Ex2 37-53 C CC G G G C C G G C A G T G G A CI108 5′ HLA-A Ex2 149-167 G T G G A T A GA G C A G G A G G G CI109 3′ HLA-A Ex3 80-100 A T G T A A T C C T T G CC G T C G CI110 3′ HLA-A Ex3 212-229 C A C T C C A C G C A C G T G C C ACI111 3′ HLA-A Ex3 105-123 A G C G C A G G T C C T C G T T C A CI112 3′HLA-A Ex3 71-88 C C G T C G T A G G C G T G C T G T CI113 3′ HLA-A Ex3110-128 C C A A G A G C G C A G G T C C T C CI114 5′ HLA-B Ex2 189-209 AC A C A G A T C T A C A A G A C C CI115 5′ HLA-C Ex2 179-197 G G A C C GG G A G A C A C A G A A CI116 3′ HLA-C Ex3 25-41 C C C C A G G T C G C AG C C A C CI117 3′ HLA-C EX3 183-200 T C T C A G C T G C T C C G C C G TCI118 3′ HLA-C Ex3 169-186 C T C A C G G G C C G C C T C C A CI119 5′HLA-C Ex2 221-239 C C G A G T G A A C C T G C G G A A CI120 5′ HLA-C Ex2249-268 T A C T A C A A C C A G A G C G A G CI121 5′ HLA-B & C Ex2210-228 C A C A G A C T G A C C G A G T G A CI122 5′ HLA-C Ex2 123-140 AG T C C A A G A G G G G A G C C G CI123 5′ HLA-A & C Ex2 5-25 C C A C TC C A T G A G G T A T T T CI124 3′ HLA-B & C Ex3 195-213 C C T C C A G GT A G G C T C T C C CI125 3′ HLA-C Ex4 234-251 C A G C C C C T C G T G CT G C A T CI126 3′ HLA-C Ex3 258-275 C G C G C G C T G C A G C G T C T TCI127 3′ HLA-C Ex3 195-213 C C T C C A G G T A G G C T C T C A CI128 3′HLA-C Ex3 18-36 G G T C G C A G C C A A A C A T C C CI129 3′ HLA-C Ex3246-265 A G C G T C T C C T T C C C A T T C CI130 5′ HLA-B Ex2 219-236 TA C C G A G A G A A C C T G C G C CI131 3′ HLA-B & C Ex3 76-93 C C T T GC C G T C G T A G G C G A CI132 3′ HLA-B Ex3 69-86 G T C G T A G G C G TC C T G G T C CI133 3′ HLA-A Ex3 20-39 C C A C G T C G C A G C C A T A CA CI134 5′ HLA-B & C Ex2 117-133MM G C C G C G A G T T C G A G A G GCI135 5′ HLA-B Ex2 220-238 A C C G A G A G A A C C T G C G G A CI136 3′HLA-A Ex2 186-205 G C C T T C A C A T T C C G T G T G CI137 3′ HLA-A Ex3216-232 A G C C C G T C C A C G C A C C G CI138 5′ HLA-A Ex2 5-25 C C AC T C C A T G A G G T A T T T CI139 5′ HLA-B Ex2 230-246 C C T G C G C AC C G C G C T C C CI140 3′ HLA-A & B 224-262 C T C T G G T T G T A G T AG C G G CI141 5′ HLA-A Ex3 63-80 G G G T A C C G G C A G G A C G C TCI142 5′ HLA-A Ex2 191-209 A C G G A A A G T G A A G G C C C A CI143 ?HLA-A Ex2 184-203 C T T C A C A T T C C G T G T C T C CI144 5′ HLA-A Ex289-107 C A C G C A G T T C G T G C G G T T CI145 3′ HLA-A Ex2 226-43 G CA G G G T C C C C A G G T C C A CI146 3′ HLA-B G C T C T G G T T G T A GT A G C G CI147 5′ HLA-B G A C G A C A C G C T G T T C G T G CI148 5′Internal Control T G C C A A G T G G A G C A C C C A CI149 3′ InternalControl G C A T C T T G C T C T G T G C A G CI150 5′ HLA-C Ex2 5-23 A CG T C G C A G C C G T A C A T G C2F30T 5′ HLA-C Ex 2 12-30 T C C A T G AA G T A T T T C A C A C2F32T 5′ HLA-C Ex2 14-32 C A T G A G G T A T T TC T A C A C C2F25A 5′ HLA-C Ex2 5-25 C A C T C C A T G A G G T A T T T CC2F25C 5′ HLA-C Ex 5-25 C A C T C C A T G A G G T A T T T C C2F32C 5′HLA-C Ex2 14-32 T G A G G T A T T T C T A C A C C G C3R195G 3′ HLA-C Ex3195-213 C C T C C A G G T A G G C T C T G T C3R195C 3′ HLA-C Ex3 195-213C T C C A G G T A G G C T C T C C G C3R076A 3′ HLA-C Ex3 76-93 C C T T GC C G T C G T A G G C G T C3R076C 3′ HLA-C Ex3 76-93 C C T T G C C G T CG T A G G C G G C3R076T 3′ HLA-C Ex3 76-93 C C T T G C C G T C G T A G GC G A C3R075TA 3′ HLA-C Ex3 75-93 C C T T G C C G T C G T A G G C T AC2F216A 5′ HLA-C Ex2 198-216 T A C A A 3 C G C C A G G C A C A G 1 2 3 45 6 7 8 9 10 11 12 13 14 15 16 17 18 CICptA1 Class I Capture Oligo A1 AC G C C T A C G A C G G C A A G G CICptA2 Class I Capture Oligo A2 G A TG G A G C C G C G G T G G A T CICptB1 Class I Capture Oligo B1 C A G T TC G T G A G G T T C G A C CICptB2 Class I Capture Oligo B2 C T G C G C GG C T A C T A C A A C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Primer 19 20 21 22 23 24 25 26 27 28 29 30 MER CI01 5′ HLA-C ex2 221-239A 19 CI02 5′ HLA-C Ex2 249-268 G A 20 CI03 5′ HLA B & C Ex2 210-228 G 19CI04 5′ HLA-C Ex2 123-140 18 CI05 5′ HLA-A & C Ex2 5-25 C T 20 CI06 3′HLA-C Ex3 243-263 A T 20 CI07 3′ HLA-C Ex3 243-263 A 19 CI08 3′ HLA-B &C Ex3 195-213 A 19 CI09 3′ HLA-C Ex4 234-251 18 CI10 3′ HLA-C Ex3258-275 18 CI11 3′ HLA-C Ex3 195-213 G 19 CI12 3′ HLA-C Ex4 31-49 18CI13 3′ HLA-C Ex3 134-151 18 CI14 3′ HLA-B & C Ex3 18-36 A 19 CI15 5′HLA-B & C Ex3 59-76 18 CI16 3′ HLA-C Ex4 4-23 T 19 CI17 3′ HLA-C Ex44-23 T T 20 CI18 5′ HLA-C Ex3 134-151 A 19 CI19 3′ HLA-C Ex3 25-42 18CI20 5′ HLA-C Ex2 195-213 18 CI21 3′ HLA-ABC Ex3 216-233 18 CI22 3′HLA-A & C Ex 3 196-214 T 19 CI23 3′ HLA-B & C Ex3 65-84 T G 20 CI24 3′HLA-C Ex3 131-148 17 CI25 5′ HLA-C Ex2 252-270 A 19 CI26 5′ HLA-C Ex2253-270 18 CI27 5′ HLA-C Ex2 85-103 A 19 CI28 3′ HLA-C Ex2 229-246 17CI29 3′ HLA-A Ex3 216-233 18 CI30 3′ HLA-ABC Ex3 216-233 18 CI31 3′HLA-A Ex3 195-213 G 19 CI32 3′ HLA-A Ex3 48-64 17 CI33 5′ HLA-A Ex2 5-25C T T 21 CI34 5′ HLA-A Ex2 168-186 C 19 CI35 3′ HLA-C Ex3 25-41 17 CI363′ HLA-B & C Ex3 169-185 17 CI37 5′ HLA-B Ex2 144-161 18 CI38 5′ HLA-BEx2 117-133 17 CI39 5′ HLA-B Ex2 181-199 G 19 CI40 5′ HLA-B Ex2 181-199C 19 CI41 5′ HLA-A & B Ex2 170-188 C 19 CI42 5′ HLA-B Ex2 195-212 18CI43 5′ HLA-B Ex2 180-199 T T 20 CI44 3′ HLA-B Ex2 219-236 18 CI45 5′HLA-B Ex2 157-173 17 CI46 5′ HLA-B Ex2 51-68 18 CI47 5′ HLA-B Ex2192-210 G 19 CI48 5′ HLA-B Ex2 5-30 C C G 21 CI49 5′ HLA-B Ex2 180-199 TA 20 CI50 5′ HLA-B & C Ex2 221-238 18 CI51 5′ HLA-A & B Ex2 220-238 T 19CI52 5′ HLA-B Ex2 116-133 18 CI53 5′ Control Primer PIC1 G G G 21 CI543′ Control Primer PICA G T T G C 23 CI55 3′ HLA-B Ex3 195-213 C 19 CI563′ HLA-B & C Ex3 44-59 16 CI57 3′ HLA-ABC Ex3 76-92 17 CI58 3′ HLA-B & CEx3 77-95 T 19 CI59 3′ HLA-B Ex3 92-111 C T 20 CI60 3′ HLA-B Ex3 201-21818 CI61 3′ HLA-ABC Ex3 216-233 18 CI62 3′ HLA-B Ex3 229-246 18 CI63 3′HLA-B Ex3 260-276 17 CI64 3′ HLA-B Ex3 262-279 18 CI65 3′ HLA-B & C Ex310-29 G A 20 CI66 3′ HLA-B Ex3 18-36 C 19 CI67 3′ HLA-B Ex3 184-201 18CI68 3′ HLA-B & C Ex3 69-87 18 CI69 3′ HLA-A & B Ex3 68-85 18 CI70 3′HLA-B Ex3 156-173 18 CI71 3′ HLA-B Ex2 173-192 A T 20 CI72 3′ HLA-A & BEx2 246-264 G 19 CI73 3′ HLA-B Ex4 168-187 C T 20 CI74 3′ HLA-B Ex311-28 18 CI75 3′ HLA-B Ex2 229-245 17 CI76 3′ HLA-ABC Ex3 216-233 18CI77 5′ HLA-A Ex3 63-80 18 CI78 5′ HLA-B & C Ex2 187-205 C G 20 CI79 3′HLA-B Ex3 120-136 17 CI80 5′ HLA-B Ex2 222-239 18 CI81 5′ HLA-B Ex2119-136 18 CI82 3′ HLA-A & B Ex3 228-245 18 CI83 5′ HLA-B Ex2 5-24 C C20 CI84 3′ HLA-B Ex3 120-136 17 CI85 3′ HLA-A & B Ex3 195-213 A 19 CI863′ HLA-B Ex2 226-243 18 CI87 5′ HLA-B Ex2 244-227 18 CI88 5′ HLA-B & CEx2 52-69 18 CI89 5′ HLA-B Ex2 116-133 18 CI90 3′ HLA-ABC Ex3 156-172 17CI91 3′ HLA-B Ex3 44-60 17 CI92 5′ HLA-B Ex2 227-244 18 CI93 5′ HLA-BEx2 222-239 18 CI94 5′ HLA-B Ex2 76-94 T 19 CI95 3′ HLA-B Ex2 207-225 T19 CI96 3′ HLA-B Ex2 207-226 T T 20 CI97 5′ HLA-A Ex2 174-192 G 19 CI983′ HLA-B & C EX3 69-87 C 19 CI99 5′ HLA-A Ex2 113-130 18 CI100 3′HLA-ABC Ex3 216-233 18 CI101 5′ HLA-A Ex2 210-229 A A 20 CI102 5′ HLA-AEx2 191-209 G 19 CI103 5′ HLA-A Ex2 111-127 17 CI104 5′ HLA-A Ex2166-184 A 19 CI105 5′ HLA-A Ex2 152-170 T 19 CI106 5′ HLA-A & B Ex2210-229 A G 20 CI107 5′ HLA-A Ex2 37-53 17 CI108 5′ HLA-A Ex2 149-167 T19 CI109 3′ HLA-A Ex3 80-100 T A A 21 CI110 3′ HLA-A Ex3 212-229 18CI111 3′ HLA-A Ex3 105-123 A 19 CI112 3′ HLA-A Ex3 71-88 18 CI113 3′HLA-A Ex3 110-128 T 19 CI114 5′ HLA-B Ex2 189-209 A A C 21 CI115 5′HLA-C Ex2 179-197 C 19 CI116 3′ HLA-C Ex3 25-41 17 CI117 3′ HLA-C EX3183-200 18 CI118 3′ HLA-C Ex3 169-186 17 CI119 5′ HLA-C Ex2 221-239 A 19CI120 5′ HLA-C Ex2 249-268 G A 20 CI121 5′ HLA-B & C Ex2 210-228 G 19CI122 5′ HLA-C Ex2 123-140 18 CI123 5′ HLA-A & C Ex2 5-25 C T C 21 CI1243′ HLA-B & C Ex3 195-213 A 19 CI125 3′ HLA-C Ex4 234-251 18 CI126 3′HLA-C Ex3 258-275 18 CI127 3′ HLA-C Ex3 195-213 G 19 CI128 3′ HLA-C Ex318-36 A 19 CI129 3′ HLA-C Ex3 246-265 T T 20 CI130 5′ HLA-B Ex2 219-236A 19 CI131 3′ HLA-B & C Ex3 76-93 18 CI132 3′ HLA-B Ex3 69-86 18 CI1333′ HLA-A Ex3 20-39 T T 20 CI134 5′ HLA-B & C Ex2 117-133MM 17 CI135 5′HLA-B Ex2 220-238 T 19 CI136 3′ HLA-A Ex2 186-205 T T 20 CI137 3′ HLA-AEx3 216-232 17 CI138 5′ HLA-A Ex2 5-25 C A C 21 CI139 5′ HLA-B Ex2230-246 17 CI140 3′ HLA-A & B 224-262 A 19 CI141 5′ HLA-A Ex3 63-80 18CI142 5′ HLA-A Ex2 191-209 G 19 CI143 ? HLA-A Ex2 184-203 C T 20 CI1445′ HLA-A Ex2 89-107 T 19 CI145 3′ HLA-A Ex2 226-43 18 CI146 3′ HLA-B G A20 CI147 5′ HLA-B A 19 CI148 5′ Internal Control A 19 CI149 3′ InternalControl A T 20 CI150 5′ HLA-C Ex2 5-23 18 C2F30T 5′ HLA-C Ex 2 12-30 T19 C2F32T 5′ HLA-C Ex2 14-32 C G C T 22 C2F25A 5′ HLA-C Ex2 5-25 G A 20C2F25C 5′ HLA-C Ex 5-25 T C 20 C2F32C 5′ HLA-C Ex2 14-32 C C 20 C3R195G3′ HLA-C Ex3 195-213 C 19 C3R195C 3′ HLA-C Ex3 195-213 18 C3R076A 3′HLA-C Ex3 76-93 18 C3R076C 3′ HLA-C Ex3 76-93 18 C3R076T 3′ HLA-C Ex376-93 18 C3R075TA 3′ HLA-C Ex3 75-93 18 C2F216A 5′ HLA-C Ex2 198-216 A19 19 20 21 22 23 24 25 26 27 28 29 30 CICptA1 Class I Capture Oligo A1A T T A C A T C G C C C CICptA2 Class I Capture Oligo A2 A G A G C A G GA G G G CICptB1 Class I Capture Oligo B1 A G C G A C G C C CICptB2 ClassI Capture Oligo B2 C A G A G C G A G G C C 19 20 21 22 23 24 25 26 27 2829 30

[0193] TABLE 2 PRIMER SEQUENCE (5′-3′) DQ01 5′ DQB 8V-1 T C C [CT] C G CA G A G G A T T T C G T DQ02 5′ DQB 26G-1 G G A G C G C G T G C G G G GDQ03 5′ DQB 26La-1 A C G G A G C G C G T G C G T C T DQ04 3′ DQB 26Y-2 GG A C G G A G C G C G T G C G T T A DQ05 3′ DQB 30H-1R G T A C T C C T CT C G G T T A T A G DQ06 3′ DQB 30S-1R G A T C T C T T C T C G G T T A TA G DQ07 3′ DQB 38V-2R G T C G C T G T C G A A G C G C A DQ08 5′ DQB55P-1 T G A C G C C G C T G G G G C C DQ09 3′ DQB 57D-2R G C T G T T C CA G T A C T C G G C G DQ10 3′ DQB 57S-2R G C T G T T C C A G T A C T C GG C G DQ11 3′ DQB 57V-1R G C T G T T C C A G T A C T C G G C A DQ12 3′DQB 70R-3R C A A C T C C G C C C G G G T C C T DQ13 5′ DQB 71K-1 G A A GG A C A T C C T G G A G A G G DQ14 3′ DQB84Q-2R G G T C G T G C G G A GC T C C A A C DQ15 3′ DQB 89G-2R C A C T C T C C T C T G C A G G A T CDQCPT1 C A C G T C G C T G T C G A A G C G C DQCPT2 C A C G T C G C T GT C G A A G C G G DQCPT3 C A C G T C G C T G T C G A A G C G T DQCPT4 CA C G T C G C T G T C G A A G C G C DQCPTS C A C G T C G C T G T C G A AG C G C DR01 5′ DR2S9-4 C C C C [AC] C A G C A C G T T T C T T G DR02 5′DR2S10G C C A G C A C G T T T C T T G DR03 5′ DR2S10L-1 [AC] C A G C A CG T T T C T T G DR04 5′ DR2S11D-2 C A C G T T T C T T G DR05 5′DR2S11R-1 C A C G T T T C T T G DR06 5′ DR2S13C-2 C G T T T C T T G G AG C A G G C T DR07 5′ DR2S13G-1 C G T T T C T T G G A G T A C T C T DR085′ DR2S13H-2 A C G T T T C T T G G A G C A G G T T DR09 5′ DR2S13R-1 C GT T T C C T G T G G C A G C C T DR10 5′ DR2S13S-2 C G T T T C T T G G AG T A C T C T DR11 5′ DR2S14K-2 C G T T T C C T G T G G C A G G G T DR123′ DR2R17-1R G T T A T G G A A DR13 5′ DR2S26L-3 C G G A G C G G G T G CG G T T G DR14 5′ DR2S26L-4 A C G G A G C G G G T G C G G T T G DR15 3′DR2R30H-1R A C T C C T C C T G G T T A T A G A A DR16 3′ DR2R37D-1R G CT G T C G A A G C G C A DR17 3′ DR2R37F-2R T C G C T G T C G A A G C G CA DR18 3′ DR2R37L-1R G C T G T C G A A G C G C A DR19 3′ DR2R37N-2R C GC T G T C G A A G C G C A DR20 3′ DR2R37S-1R G C T G T C G A A G C G C ADR21 3′ DR2R37Y-1R G C T G T C G A A G C G C A DR22 5′ DR2S37YA-1 C G CT G T C G T A G C G C G C G T DR23 3′ DR2R47F-2R T C C G T C A C C G C CC G G A DR24 5′ DR2S52B-3 G G A G T A C C G G G C G G T G A G DR25 3′DR2R57D-1R C T G T T C C A G T A C T C G G C DR26 3′ DR2R57S-1R T G T TC C A G T A C T C G G C DR27 3′ DR2R57V-1R C T G T T C C A G G A C T C GG C DR28 3′ DR2R58E-1R T C A G G C T G T T C C A G T A C T DR29 3′DR2R67F-2R C G C G C C T G T C T T C C A G G A A DR30 3′ DR2R67I-2R C CC G C T C G T C T T C C A G G A T DR31 3′ DR2R70QR-3 C A C C G C G G C CC G C C T C T G DR32 3′ DR2R71A-2R C A C C G C G G C C C G C G C DR33 3′DR2R74E-1R T G C A A T A G G T G T C C DR34 3′ DR2R74L-1R T G C A G T AG G T G T C C DR35 3′ DR2R74Q-2R G T G T C T G C A G T A A T T G T C CDR36 3′ DR2R74R-1R G T G T C T G C A G T A A T T G T C C DR37 3′DR2R76G-1R A T G T C T G C A DR38 3′ DR2R81Y-1R C T C T C C A C C A A CC C G T A G T DR39 3′ DR2R86G-1R T G C A C T G T G A A G C T C T C ADR40 3′ DR2R86V-1R C T G C A C T G T G A A G C T C T C C DR41 3′DR2R78A-1R C C C C G T A G T T G T G T C T G C A DR42 3′ DR2R74C-IR G CA G T A G G T G T C C DR43 3′ DR2R74T-1R G C A A T A G G T G T C C DR443′ DR2R60T-1R C C T T C T G G C T G T T C C A G T DR45 3′ DR2R60G-1R T CC T T C T G G C T G T T C C A G G DR46 3′ DR2R85A-1R A C A G T G A A G CT C T C C DR47 3′ DR2R47F C T C C G T C A C C G C C C G G A DR48 3′DR2R477-1R C T C C G T C A C C G C C C G G T A DR49 3′ DR2R30a C T C C TC C T G G T T A T G G A A DR50 3′ DR2R30b C T C C T C C T G G T T A T GG A A DR51 3′ DR2R37a T C G C T G T C G A A G C G C A DR52 3′ DR2R37b CG C T G T C G A A G C G C A DR53 3′ DR2R37c C G C T G T C G A A G C G CA DR54 3′ DR2R37d T C G C T G T C G A A G C G C A DR55 3′ DR2R37e T C GC T G T C G A A G C G C A DR56 3′ DR2R38a A C G T C G C T G T C G A A GC G C A DR57 3′ DR2R45a T C A C C G C C C G G T A C DR58 3^(′) DR2R48a CC A G C T C C G T C A C C G C C T DR59 3′ DR2R50a C C G C C C C A G C TC C G T C G DR60 3′ DR2R57a G C Y G T T C C A G T G C T C C G C DR61 3′DR2R57b G C T G T T C C A G T G C T C C G C DR62 3′ DR2R57c G G C T G TT C C A G T A C T C A G C DR63 3′ DR2R57c2 G C T G T T C C A G T A C T CG G C DR64 3′ DR2R58a T T C T G G C T G T T C C A G T A C T DR65 3′DR2R67a C C G C C T C T G C T C C A G G A G DR66 3′ DR2R67b C C G C G CC T G C T C C A G G A T DR67 3′ DR2R69a A C C G C G G C G C G C C T G TC T DR68 3′ DR2R69b C C G C G G C C C G C G C C T G C DR69 3′ DR2R70a CA C C G C G G C G C G C C T G T T DR70 3′ DR2R70b C A C C T C G G C C CG C C T C C DR71 3′ DR2R71a G T C C A C C G C G G C G C G C G T DR72 3′DR2R71b T G T C C A C C G C G G C C C G C T DR73 3′ DR2R71c T C C A C CG C G G C C C G C G C DR74 3′ DR2R71c2 T C C A C C G C G G C C C G C T CDR75 3′ DR2R71d T G T C C A C C G C G G C C C G C T DR76 3′ DR2R72a T AG G T G T C C A C C G C G G C G DR77 3′ DR2R72b G C G C C A C C T G T GG A T G A C G DR78 3′ DR2R74b T C T G C A G T A A T T G T C C DR79 3′DR2R74a G T C T G C A A T A G G T G T C C DR80 3′ DR2R74c C T G C A G TA G T T G T C C DR81 3′ DR2R77a C C G T A G T T G T A T C T G C A DR823′ DR2R77b C C G T A G T T G T G T C T G C A DR83 3′ DR2R77b C C C G T AG T T G T G T C T G C A DR84 3′ DR2R78a C C C G T A G T T G T G T C T GC A DR85 5′ DR2S11A C A G C A C G T T T C T T G DR86 5′ DR2S14b T T C TT G T G G C A G C T T DRCPT1 DRCPTA T G G C G T G G G C G A G G C A G GG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 5′ DPA - E (PC) G A TC C C C C T G A G G T G A C C G 3′ DPA - F (PC) C T G G G C C C G G G GG T C A T G G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 PRIMERSEQUENCE (5′-3′) MER 3′ seq DQ01 5′ DQB 8V-1 G 20 G DQ02 5′ DQB 26G-1 15G DQ03 5′ DQB 26La-1 17 T DQ04 3′ DQB 26Y-2 19 A DQ05 3′ DQB 30H-1R A TG T G 24 C DQ06 3′ DQB 30S-1R A T G C 23 G DQ07 3′ DQB 38V-2R 17 T DQ085′ DQB 55P-1 16 G DQ09 3′ DQB 57D-2R T 20 A DQ10 3′ DQB 57S-2R C T 21 ADQ11 3′ DQB 57V-1R A 20 T DQ12 3′ DQB 70R-3R 18 A DQ13 5′ DQB 71K-1 A A21 A DQ14 3′ DQB84Q-2R T G 21 C DQ15 3′ DQB 89G-2R C C 21 G DQCPT1 A C GT A C T C C T C 30 C DQCPT2 A C G A T C T C C T T 30 T DQCPT3 G C G T AC T C C T C 30 C DQCPT4 G C G T A C T C C T C 30 C DQCPTS A C G T C C TC C T C 30 C DR01 5′ DR2S9-4 A 20 A DR02 5′ DR2S10G G A G G 19 G DR03 5′DR2S10L-1 G A G C T 20 T DR04 5′ DR2S11D-2 C A G C A G G A 19 A DR05 5′DR2S11R-1 G A G C T G C G 19 G DR06 5′ DR2S13C-2 A A G T G 23 G DR07 5′DR2S13G-1 A C G G G 23 G DR08 5′ DR2S13H-2 A A A C 23 C DR09 5′DR2S13R-1 A A G A 22 A DR10 5′ DR2S13S-2 A C G T C 23 C DR11 5′DR2S14K-2 A A G T A T A 25 A DR12 3′ DR2R17-1R G T A T C T G T C C A G GT 23 A DR13 5′ DR2S26L-3 17 G DR14 5′ DR2S26L-4 18 G DR15 3′ DR2R30H-1RG T G 22 C DR16 3′ DR2R37D-1R A G T C 18 G DR17 3′ DR2R37F-2R C G A 19 TDR18 3′ DR2R37L-1R G G A G 18 C DR19 3′ DR2R37N-2R C G T T 19 A DR20 3′DR2R37S-1R C G G 17 C DR21 3′ DR2R37Y-1R C G T A 18 T DR22 5′ DR2S37YA-118 A DR23 3′ DR2R47F-2R 16 T DR24 5′ DR2S52B-3 18 G DR25 3′ DR2R57D-1R AT 19 A DR26 3′ DR2R57S-1R G C T 19 A DR27 3′ DR2R57V-1R G A 21 T DR28 3′DR2R58E-1R C C T 21 A DR29 3′ DR2R67F-2R 19 T DR30 3′ DR2R67I-2R 19 ADR31 3′ DR2R70QR-3 18 C DR32 3′ DR2R71A-2R 15 G DR33 3′ DR2R74E-1R A C CT C 19 G DR34 3′ DR2R74L-1R A C C A G 19 C DR35 3′ DR2R74Q-2R A C C T G24 C DR36 3′ DR2R74R-1R A C C C 23 G DR37 3′ DR2R76G-1R G T A G G T G C17 G DR38 3′ DR2R81Y-1R T G T A 23 T DR39 3′ DR2R86G-1R C 19 G DR40 3′DR2R86V-1R A 20 T DR41 3′ DR2R78A-1R A 20 T DR42 3′ DR2R74C-IR A C C G C18 G DR43 3′ DR2R74T-1R A C C T C 18 G DR44 3′ DR2R60T-1R G 19 C DR45 3′DR2R60G-1R 19 C DR46 3′ DR2R85A-1R A C A G 19 C DR47 3′ DR2R47F 17 TDR48 3′ DR2R477-1R 18 T DR49 3′ DR2R30a G T G 21 C DR50 3′ DR2R30b G T A21 T DR51 3′ DR2R37a C G T C 20 G DR52 3′ DR2R37b C G G A 19 T DR53 3′DR2R37c C G T C 19 G DR54 3′ DR2R37d G G A 19 T DR55 3′ DR2R37e C G A 19T DR56 3′ DR2R38a G 20 C DR57 3′ DR2R45a T C C C T 19 A DR58 3^(′)DR2R48a 18 A DR59 3′ DR2R50a 17 C DR60 3′ DR2R57a A G 20 C DR61 3′DR2R57b A T 20 A DR62 3′ DR2R57c G 20 C DR63 3′ DR2R57c2 G A T 21 A DR643′ DR2R58a C A 21 T DR65 3′ DR2R67a 19 C DR66 3′ DR2R67b 18 A DR67 3′DR2R69a 18 A DR68 3′ DR2R69b 17 G DR69 3′ DR2R70a 18 A DR70 3′ DR2R70b17 G DR71 3′ DR2R71a 18 A DR72 3′ DR2R71b 18 A DR73 3′ DR2R71c 17 G DR743′ DR2R71c2 17 G DR75 3′ DR2R71d 17 A DR76 3′ DR2R72a 18 C DR77 3′DR2R72b 19 C DR78 3′ DR2R74b A C C T G 21 C DR79 3′ DR2R74a A C C T 21 ADR80 3′ DR2R74c A C C C G 20 C DR81 3′ DR2R77a G T A G T 22 A DR82 3′DR2R77b G T A G T 22 A DR83 3′ DR2R77b G T A A T 23 A DR84 3′ DR2R78a CA C 21 G DR85 5′ DR2S11A G A G C T G T 21 T DR86 5′ DR2S14b A A G T T TG A A 24 A DRCPT1 DRCPTA T A A C T T C T T T A 1 T 20 21 22 23 24 25 2627 28 29 30 5′ DPA - E (PC) T G 21 G 3′ DPA - F (PC) C C 21 G 20 21 2223 24 25 26 27 28 29 30 MER

[0194] TABLE 3 Size 5′ Primer 3′ Primer Specificity (bp) A01 1 CI099CI137 A*0101, 0102 629 A02 4 CI099 CI030 A*3601 630 A03 2 CI108 CI113A*0201-17 489 A04 3 CI103 CI110 A*0301, 0302 628 A05 15 CI102 CI029A*1101, 1102, 552 6601 A06 6 CI104 CI085 A*2301 557 A07 5 CI097 CI113A*2301, 464 A*2401-07 A08 7 CI104 CI031 A*2402-05, 557 2407 A09 10 CI106CI109 A*2501 400 A10 8 CI077 CI029, 021 A*2501, 2601, 170 2603, 2605,6601, 6602, 4301 A11 9 CI041 CI109 A*2501, 440 2601-05, 6601, 6602,3401, 3402 A12 11 CI101 CI109 A*2601, 2602, 400 2604, 4301 B01 12 CI034CI109 A*4301 442 B02 13 CI077, 141 CI030 A*3401, 3402 170 B03 14 CI102,142 CI109 A*3401, 3402, 419 6601, 6602 B04 16 CI034 CI111 A*2901, 2902465 B05 17 CI107 CI112 A*3001-05 561 B06 18 CI138 CI143 A*3101 198 B0719 CI033 CI072 A*3201 259 B08 21 CI033 CI111 A*3201, 7401 628 B09 20CI138 CI136 A*3301-03 200 B10 22 CI102 CI113 A*6801, 6802, 447 6901 B1123 CI102 CI032 A*6901 383 B12 24 CI120 CI100 A*8001 494 C01 25 CI120CI133 A*01, *11, 300 *3601, *3401, *8001 C02 79 CI051 CI059 B*5101-05,401 51v, 51GAC, 5201 C03 80 CI041 CI059 B*5101-05, 451 51v, 51GAC,7801-02, 1509 C04 81 CI040 CI059 B*5201 440 C05 77 CI043 CI056, 091B*3501-09, 389/340 3511, 5301 C06 28 CI041 CI064 B*0702-05, 619 8101 C0729 CI114 CI064 B*0703 600 C08 30 CI043 CI055 B*0801, 0802, 543 B51GAC,B*4406 C09 31 CI043 CI063 B*0801, 0802 606 C10 36 CI046, 089 CI132, 098B*4402-06 546/481 C11 34 CI083 CI058 B*4501, 45v, 600 4901, 5001 C12 35CI050 CI062 B*4501, 45V, 536 1514 D01 42 CI081 CI058 B*1301-03 486 D0243 CI045 CI014 B*1401, 1402 389 D03 44 CI048 CI071 B*1402, 3904 187 D0467 CI081 CI086 B*1501, 1502, 124 1504-08, 1511, 1512, 1514, 1515,1519-21, 1525, 1526N, 1528 D05 68 CI040 CI057 B*1501, 1503-07, 421 1512,1514, 1519, 1520, 1524, 1525, 4802, 4003, 13x15, 1526N D06 70 CI052CI057 B*1503, 1518, 486 1523, 1529, 4802, 3907, 72v, Cw0703 D07 72 CI039CI076 B*1509, 1510, 562 1518, 1521, 1523 D08 73 CI081 CI062, 082 B*1512,1514, 636/637 1519 D09 74 CI041 CI124 B*1508, 1511, 553 1515, 1522,A*68, 2501, 2601-05, 3401, 6601-02 D10 65 CI042 CI067 B*1516, 1517 516D11 47 CI051, 139 CI060 B*3801, 3802 498/508 D12 48 CI052 CI060 B*3801,3802, 612 3901-08, 6701 E01 45 CI050 CI060 B*3901-08, 6701 507 E02 46CI049 CI060 B*6701 548 E03 51 CI042 CI066 B*5701-03 351 E04 52 CI081CI140 B*5701-03, 1513, 143 1516, 1517, 1524, 1301-03, 13x15 E05 50 CI042CI056 B*5801-03 374 E06 49 CI051 CI065 B*5801, 5104, 319 5301, 1513 E0753 CI037 CI057 B*1801, 1802 458 E08 41 CI094 CI070 B*4001, 4007 607 E0940 CI089 CI061 B*4001-04, 627 4006-08, 4701 E10 38 CI089 CI090B*4002-06, 4008, 566 4101, 4102, 4501, 45v, 4901, 5001, 4402-05, 4701E11 33 CI051 CI058 B*4901, 5901 385 E12 32 CI094 CI067 B*4901, 5001, 6354005, 2704, 2706, 45v F01 57 CI134 CI074 B*5401 421 F02 55 CI080 CI058B*5401, 5501, 383 5502, 5601, 4501, 45v, 5001 F03 54 CI052 CI074 B*5501,5502, 422 5601, 5602, 7301, 3906 F04 56 CI047 CI076 B*5601, 5602 551 F0558 CI094 CI095, 096 B*2701-09 149/150 F06 75 CI041 CI065 B*3501-04, 3693506-09, 3511, 5301, 1502, 1513, 5104, 1521, 4406 F07 76 CI038 CI075B*3501-13, 18, 128 7801-02, 1522 F08 59 CI038 CI055 B*3701, B*4406, 606B51GAC F09 60 CI040 CI131 B*3701, 3902, 422 3908 F10 37 CI040 CI063B*4101, 4102 605 F11 63 CI047 CI063 B*4201, 42v 594 F12 66 CI078 CI079B*4601 459 G01 61 CI040 CI069 B*4701 414 G02 64 CI052 CI070 B*4801, 8101567 G03 39 CI040 CI084 B*4801, 4001-06, 465 weak B41 G04 69 CI088 CI065B*4802 487 G05 71 CI088 CI076 B*4802, 1503, 691 1509, 1510, 1518, 1523,1529, 72v G06 62 CI120 CI074 B*7301 289 G07 78 CI050 CI059 B*7801-02,1509 400 G08 26 CI051, 087, CI073 Bw4 1330 092, 139 G09 27 CI080 CI073Bw6 not B73 1340 G10 82 CI121 CI116 Cw*0101, 0102 341 G11 83 CI119 CI021Cw*0201, 0202, 522 1701 G12 84 CI121 CI129 Cw*0302, 0303, 565 0304 H0185 CI119 CI019 Cw*0401, 0402 331 H02 86 CI119 CI126 Cw*0501 564 H03 87CI120 CI014 Cw*0602 297 H04 88 CI015 CI125 Cw*0701, 0702, 1062 0703 H0589 CI115 CI036 Cw*0701 516 H06 90 CI120 CI035 Cw*0702, 0703 302 H07 91CI120 CI076 Cw*0703, A*2604 494 H08 92 CI120 CI126 CW*0704 536 H09 93CI027 CI028, 117 Cw*0802 Cw*0801/3 161/625 H10 94 CI025 CI129 Cw*0303523 H11 95 CI026 CI129 Cw*0302, 0304 522 H12 96 Neg. 0 Control

[0195] TABLE 4 Primer Primer Tray Mix S AS label A01 DRM01 DR13 DR31DR2R70QR DRB1*0102 A02 DRM02 DR13 DR20 DR2R37S DRB1*0101, 0102, 0103,0104 A03 DRM03 DR13 DR30 DR2R67I DRB1*0103 A04 DRM04 DR13 DR39 DR2R86GDRB1*0101, 0103 A05 DRM05 DR13 DR40 DR2R86V DRB1*0102, 0104 A06 DRM06DR02 DR39 DR2R86G DRB1*1001 A07 DRM07 DR02 DR25 DR2R57D DRB1*1001 A08DRM08 DR09 DR15 DR2R30H DRB1*1503 A09 DRM09 DR09 DR17 DR2R37F DRB1*1608A10 DRM10 DR09 DR23 DR2R47F DRB1*1501, 1502, 1503, 1504, 1505, 1506,1508, 1510 A11 DRM11 DR09 DR48 DR2R47? DRB1*1507, 16XX A12 DRM12 DR09DR25 DR2R57D DRB1*1502 B01 DRM13 DR09 DR30 DR2R67I DRB1*1510, 1605, 1607B02 DRM14 DR09 DR29 DR2R67F DRB1*1601, 1603?, 1604 B03 DRM15 DR09 DR32DR2R71A DRB1*15XX B04 DRM16 DR09 DR34 DR2R74L DRB1*1604 B05 DRM17 DR09DR39 DR2R86G DRB1*1502, 16XX B06 DRM18 DR09 DR40 DR2R86V DRB1*1501,1503, 1504, 1505, 1506, 1507, 1509, 1510 B07 DRM19 DR10 DR12 DR17-1RDRB1*0301, 0304, 5, 6, 8-16 B08 DRM20 DR10 DR21 DR2R37Y DRB1*11XX, 1303,07, 11-14, 17, 21-25, 30, 33, 37, 38, 44, 45, 1425 B09 DRM21 DR10 DR19DR2R37N DRB1*0301, 02, 05-15, 1109, 16, 20, 28, 1301, 02, 05, 06, 09,10, 15, 16, 18, 20, 26-29, 31, 32, 34-36, 39-43, 1402, 03, 06, 09, 12,13, 17- 19, 21, 24, 27, 29, 30, 33 B10 DRM22 DR10 DR17 DR2R37FDRB1*1110, 12, 13, 17, 1308, 19, 1401, 04, 05, 07, 08, 10, 11, 14-16,20, 22, 23, 26, 28, 31, 32, 34-36 B11 DRM23 DR10 DR23 DR2R47F DRB1*0301,04, 05, 07-14, 1101-16, 18-36, 38, 39, 1301, 02, 04-06, 14-18, 20-25,27-31, 34, 35?, 39, 41-45, 1417, 21, 30, 33, 35, B12 DRM24 DR10 DR48DR2R47? DRB1*0302, 03, 06, 1117, 37, 1303, 07, 08, 12, 13, 19, 26, 32,33, 36-38, 40, 1401-16, 18-20, 22-29, 31, 32, 34, 36 C01 DRM25 DR10 DR25DR2R57D DRB1*0301-07, 11, 13-16, 1301, 02, 05-11, 14-20, 22-25, 27-29,34-37, 39-42, 44, 1402, 03, 06, 09, 12, 14, 15, 17-21, 23, 24, 27, 29,30, 33, 36 C02 DRM26 DR10 DR26 DR2R57S DRB1*0312, 1303, 04, 12, 13, 21,30, 32, 33, 38, 1413, C03 DRM27 DR10 DR27 DR2R57V DRB1*1331 C04 DRM28DR10 DR28 DR2R58E DRB1*11XX, 1411, C05 DRM29 DR10 DR29 DR2R67FDRB1*1101, 03-06, 09-12, 15, 22-25, 27-30, 32, 33, 35, 37-39, 1305, 07,11, 14, 18, 21, 24, 26, 42, 1415, 22, 25, 27 C06 DRM30 DR10 DR30 DR2R67IDRB1*1102, 14, 16, 20, 21, 1301-04, 08, 10, 15, 16, 1922, 23, 27, 28,31-41, 45, 1416 C07 DRM31 DR10 DR31 DR2R70QR DRB1*1126, 34, 1344, 1402,06, 09, 13, 17, 20, 29, 30, 33 C08 DRM32 DR10 DR34 DR2R74L DRB1*0820,1123, 25, 1313, 18, 1403, 12, 27 C09 DRM33 DR10 DR46 DR2R85? DRB1*1106,21, 1429 C10 DRM34 DR10 DR39 DR2R86G DRB1*0302, 05, 09, 14, 17, 1101,08-12, 14, 15, 19, 20, 23, 24, 26-29, 31-33, 37, 39, 1302, 03, 05, 07,12-14, 21, 23, 25, 26, 29-31, 33, 34, 36- 39, 41, 45, 1402, 03, 07, 09,13, 14, 19, 22, 24, 25, 27, 30, 36 C11 DRM35 DR10 DR40 DR2R86VDRB1*0301, 03, 04, 06-08, 10-13, 15, 16, 0820, 1102-04, 06, 07, 13,16-18, 21, 25, 34-36, 38, 1301, 04, 06, 08-11, 15, 18- 20, 22, 24, 27,28, 32, 35, 40, 42-44, 1401, 05, 06, 08, 12, 16-18, 20, 21, 23, 26, 29,32-35 C12 DRM36 DR07 DR21 DR2R37Y DRB1*0801-08, 10-15, 17-19, 1105, 1317D01 DRM37 DR07 DR18 DR2R37L DRB1*1201-04, 1206 D02 DRM38 DR07 DR23DR2R47F DRB1*0817, 1105, 1201-06, 1317 D03 DRM39 DR07 DR48 DR2R47?DRB1*0801-17, 18, 19, 21, 1404, 11, 15, 28, 31 D04 DRM40 DR07 DR26DR2R57S DRB1*0801, 03, 05, 06, 10, 12, 14, 16-19w D05 DRM41 DR07 DR25DR2R57D DRB1*0802, 04, 09, 13, 15, 21, 1105, 1204, 1317, 1411, 15 D06DRM42 DR07 DR27 DR2R57V DRB1*1201-03, 05, 06 D07 DRM43 DR07 DR28 DR2R58EDRB1*1105, 1204, 1411 D08 DRM44 DR07 DR44 DR2R60? DRB1*0808, 15, 1404,28, 31 D09 DRM45 DR07 DR45 DR2R60? DRB1*1201-03, 05, 06 D10 DRM46 DR07DR29 DR2R67F DRB1*0801, 02, 04-09, 11, 16, 17, 21, 1105, 1202, 1415 D11DRM47 DR07 DR34 DR2R74L DRB1*0801-04, 06-19, , 21, 1415 D12 DRM48 DR07DR46 DR2R85? DRB1*0812, 1201, 02, 04-06, 1428 E01 DRM49 DR07 DR39DR2R86G DRB1*0801-03, 05, 07-09, 11, 13-19, 21, 1105 E02 DRM50 DR07 DR40DR2R86V DRB1*0804, 06, 10, 12, 1201-06, 1404, 11, 15, 28, 31 E03 DRM51DR08 DR20 DR2R37S DRB1*0406, 19-21 E04 DRM52 DR08 DR21 DR2R37YDRB1*0401-05, 07-18, 22-36, 1122, 1410 E05 DRM53 DR08 DR23 DR2R47FDRB1*0428, 35, 1122 E06 DRM54 DR08 DR26 DR2R57S DRB1*0405, 09-12, 17,24, 28-30 E07 DRM55 DR08 DR25 DR2R57D DRB1*0401-04, 06-08, 13, 14, 16,18-23, 25-27, 31-36 E08 DRM56 DR08 DR28 DR2R58E DRB1*0415, 1122 E09DRM57 DR08 DR29 DR2R67F DRB1*0415, 25, 36, 1122 E10 DRM58 DR08 DR30DR2R67I DRB1*0402, 12w, 14, 18 E11 DRM59 DR08 DR70 DR2R70B DRB1*0401,09, 13, 16, 21, 22, 26, 33-35 E12 DRM60 DR08 DR33 DR2R74E DRB1*0403, 06,07, 11, 17, 20, 22, 27, 1410 F01 DRM61 DR08 DR34 DR2R74L DRB1*0412, 18,25, 31 F02 DRM62 DR08 DR39 DR2R86G DRB1*0401, 05, 07-09, 14, 16, 17,19-21, 24, 26, 28-31, 33-35, 1122 F03 DRM63 DR08 DR40 DR2R86VDRB1*0402-04, 06, 10-13, 15, 18, 22, 23, 25, 27, 32, 36, 1410 F04 DRM64DR11 DR17 DR2R37F DRB1*0701, 03, 04 F05 DRM65 DR11 DR39 DR2R86GDRB1*0701, 03, 04 F06 DRM66 DR01 DR27 DR2R57V DRB1*0901 F07 DRM67 DR01DR39 DR2R86G DRB1*0901 F08 DRM68 DR03 DR20 DR2R37S DRB3*0203 F09 DRM69DR03 DR21 DR2R37Y DRB1*1130 F10 DRM70 DR03 DR19 DR2R37N DRB3*0206? F11DRM71 DR03 DR17 DR2R37F DRB3*0301-03 F12 DRM72 DR03 DR26 DR2R57SDRB3*0208 G01 DRM73 DR03 DR25 DR2R57D DRB3*0107, 0201-06, 10-13 G02DRM74 DR03 DR35 DR2R74Q DRB3*0107, 0201-03, 05-13, 0301, 02 G03 DRM75DR03 DR36 DR2R74R DRB3*0101-06 G04 DRM76 DR03 DR39 DR2R86G DRB3*0101-07,0202, 03, 05-13, 0303 G05 DRM77 DR03 DR40 DR2R86V DRB3*0201, 04, 0301,02 G06 DRM78 DR05 DR25 DR2R57D DRB3*0107 G07 DRM79 DR05 DR39 DR2R86GDRB3*0101-07 G08 DRM80 DR06 DR37 DR2R76G DRB4*0102 G09 DRM81 DR06 DR38DR2R81Y DRB4*0101-04 G10 DRM82 DR06 DR40 DR2R86V DRB4*0101-05 G11 DRM83DR04 DR20 DR2R37S NEG G12 DRM84 DR04 DR16 DR2R37D DRB5*0101, 04-07, 09H01 DRM85 DR04 DR29 DR2R67F DRB5*0101-05, 08-10 H02 DRM86 DR04 DR32DR2R71A DRB5*0106, 0202-04 H03 DRM87 DR04 DR34 DR2R74L DRB5*0104 H04DRM88 DR04 DR39 DR2R86G DRB5*0101-05, 07-10, 0203 H05 DRM89 DR04 DR40DR2R86V DRB5*0106, 0202, 04, 05 Mix P1 P2 15.0 μl 15 μl

[0196] TABLE 5 ID Location CI06 3′ HLA-C Ex 3 243-263 Biotin CI07 3′HLA-C Ex 3 243-263 Biotin CI08 3′ HLA-B & C Ex 3 195-213 Biotin CI09 3′HLA-C Ex 4 234-251 Biotin CI10 3′ HLA-C Ex 3 258-275 Biotin CI11 3′HLA-C Ex 3 195-213 Biotin CI12 3′ HLA-C Ex 4 31-49 Biotin CI13 3′ HLA-CEx 3 134-151 Biotin CI14 3′ HLA-B & C Ex 3 18-36 Biotin CI16 3′ HLA-C Ex4 4-23 Biotin CI17 3′ HLA-C Ex 4 4-23 Biotin CI19 3′ HLA-C Ex 3 25-42Biotin CI21 3′ HLA-ABC Ex 3 216-233 Biotin CI22 3′ HLA-A & C Ex 3196-214 Biotin CI23 3′ HLA-B & C Ex 3 65-84 Biotin CI24 3′ HLA-C Ex 3131-148 Biotin CI28 3′ HLA-C Ex 2 229-246 Biotin CI29 3′ HLA-A Ex 3216-233 Biotin CI30 3′ HLA-ABC Ex 3 216-233 Biotin CI31 3′ HLA-A Ex 3195-213 Biotin CI32 3′ HLA-A Ex 3 48-64 Biotin CI35 3′ HLA-C Ex 3 25-41Biotin CI36 3′ HLA-B & C Ex 3 169-185 Biotin CI44 3′ HLA-B Ex 2 219-236Biotin CI55 3′ HLA-B Ex 3 195-213 Biotin CI56 3′ HLA-B & C Ex 3 44-59Biotin CI57 3′ HLA-ABC Ex 3 76-92 Biotin CI58 3′ HLA-B & C Ex 3 77-95Biotin CI59 3′ HLA-B Ex 3 92-111 Biotin CI60 3′ HLA-B Ex 3 201-218Biotin CI61 3′ HLA-ABC Ex 3 216-233 Biotin CI62 3′ HLA-B Ex 3 229-246Biotin CI63 3′ HLA-B Ex 3 260-276 Biotin CI64 3′ HLA-B Ex 3 262-279Biotin CI65 3′ HLA-B & C Ex 3 10-29 Biotin CI66 3′ HLA-B Ex 3 18-36Biotin CI67 3′ HLA-B Ex 3 184-201 Biotin CI68 3′ HLA-B & C Ex 3 69-87Biotin CI69 3′ HLA-A & B Ex 3 68-85 Biotin CI70 3′ HLA-B Ex 3 156-173Biotin CI71 3′ HLA-B Ex 2 173-192 Biotin CI72 3′ HLA-A & B Ex 2 246-264Biotin CI73 3′ HLA-B Ex 4 168-187 Biotin CI74 3′ HLA-B Ex 3 11-28 BiotinCI75 3′ HLA-B Ex 2 229-245 Biotin CI76 3′ HLA-ABC Ex 3 216-233 BiotinCI79 3′ HLA-B Ex 3 120-136 Biotin CI82 3′ HLA-A & B Ex 3 228-245 BiotinCI84 3′ HLA-B Ex 3 120-136 Biotin CI86 3′ HLA-B Ex 2 226-243 Biotin CI903′ HLA-ABC Ex 3 156-172 Biotin CI91 3′ HLA-B Ex 3 44-60 Biotin CI95 3′HLA-B Ex 2 207-225 Biotin CI96 3′ HLA-B Ex 2 207-226 Biotin CI98 3′HLA-B & C EX 3 69-87 Biotin CI100 3′ HLA-ABC Ex 3 216-233 Biotin CI1093′ HLA-A Ex 3 80-100 Biotin CI110 3′ HLA-A Ex 3 212-229 Biotin CI111 3′HLA-A Ex 3 105-123 Biotin CI112 3′ HLA-A Ex 3 71-88 Biotin CI113 3′HLA-A Ex 3 110-128 Biotin CI116 3′ HLA-C Ex 3 25-41 Biotin CI117 3′HLA-C EX 3 183-200 Biotin CI118 3′ HLA-C Ex 3 169-186 Biotin CI124 3′HLA-B & C Ex 3 195-213 Biotin CI125 3′ HLA-C Ex 4 234-251 Biotin CI1263′ HLA-C Ex 3 258-275 Biotin CI127 3′ HLA-C Ex 3 195-213 Biotin CI128 3′HLA-C Ex 3 18-36 Biotin CI129 3′ HLA-C Ex 3 246-265 Biotin CI131 3′HLA-B & C Ex 3 76-93 Biotin CI132 3′ HLA-B Ex 3 69-86 Biotin CI133 3′HLA-A Ex 3 20-39 Biotin CI136 3′ HLA-A Ex 2 186-205 Biotin CI137 3′HLA-A Ex 3 216-232 Biotin CI140 3′ HLA-A & B 224-262 Biotin CI143 3′HLA-A Ex 2 184-203 Biotin CI145 3′ HLA-A Ex 2 226-43 Biotin CI146 3′HLA-B Biotin CI149 3′ Internal Control Biotin C3R195G 3′ HLA-C Ex 3195-213 Biotin C3R195C 3′ HLA-C Ex 3 195-213 Biotin C3R076A 3′ HLA-C Ex3 76-93 Biotin C3R076C 3′ HLA-C Ex 3 76-93 Biotin C3R076T 3′ HLA-C Ex 376-93 Biotin C3R075TA 3′ HLA-C Ex 3 75-93 Biotin

[0197] TABLE 6 ID PRIMER DQ01 5′ Biotin DQB 8V-1 DQ02 5′ Biotin DQB26G-1 DQ03 5′ Biotin DQB 26La-1 DQ04 5′ Biotin DQB 26Y-2 DQ08 5′ BiotinDQB 55P-1 DQ13 5′ Biotin DQB 71K-1 DR01 5′ Biotin DR2S9-4 DR02 5′ BiotinDR2S10G DR03 5′ Biotin DR2S10L-1 DR04 5′ Biotin DR2S11D-2 DR05 5′ BiotinDR2S11R-1 DR06 5′ Biotin DR2S13C-2 DR07 5′ Biotin DR2S13G-1 DR08 5′Biotin DR2S13H-2 DR09 5′ Biotin DR2S13R-1 DR10 5′ Biotin DR2S13S-2 DR115′ Biotin DR2S14K-2 DR12 5′ DR2R17-1R DR13 5′ Biotin DR2S26L-3 DR14 5′Biotin DR2S26L-4 DR22 5′ Biotin DR2S37YA- 1 DR24 5′ Biotin DR2S52B-3DR85 5′ Biotin DR2S11A DR86 5′ Biotin DR2S14b 5′ Biotin DPA - E (PC)

[0198]

1 278 1 19 DNA Homo sapiens 1 ccgagtgaac ctgcggaaa 19 2 20 DNA Homosapiens 2 tactacaacc agagcgagga 20 3 19 DNA Homo sapiens 3 cacagactgaccgagtgag 19 4 18 DNA Homo sapiens 4 agtccaagag gggagccg 18 5 20 DNAHomo sapiens 5 ccactccatg aggtatttct 20 6 20 DNA Homo sapiens 6tcttctccag aaggcaccat 20 7 19 DNA Homo sapiens 7 caggtcagtg tgatctcca 198 19 DNA Homo sapiens 8 cctccaggta ggctctcca 19 9 18 DNA Homo sapiens 9cagcccctcg tgctgcat 18 10 18 DNA Homo sapiens 10 cgcgcgctgc agcgtctt 1811 19 DNA Homo sapiens 11 cctccaggta ggctctcag 19 12 18 DNA Homo sapiens12 ctcagggtga ggggctct 18 13 18 DNA Homo sapiens 13 tgagccgccg tgtccgca18 14 19 DNA Homo sapiens 14 ggtcgcagcc atacatcca 19 15 18 DNA Homosapiens 15 ccgcgggtat gaccagtc 18 16 19 DNA Homo sapiens 16 gcgtctccttcccgttctt 19 17 20 DNA Homo sapiens 17 agcgtctcct tcccattctt 20 18 19DNA Homo sapiens 18 tccgcgggta tgaccagta 19 19 18 DNA Homo sapiens 19gccccaggtc gcagccaa 18 20 18 DNA Homo sapiens 20 acaagcgcca ggcacagg 1821 18 DNA Homo sapiens 21 gagccactcc acgcactc 18 22 19 DNA Homo sapiens22 ccctccaggt aggctctct 19 23 20 DNA Homo sapiens 23 tcgtaggctaactggtcatg 20 24 17 DNA Homo sapiens 24 ccgccgtgtc cgcggca 17 25 19 DNAHomo sapiens 25 tacaaccaga gcgaggcca 19 26 18 DNA Homo sapiens 26acaaccagag cgaggccg 18 27 19 DNA Homo sapiens 27 acgacacgca gttcgtgca 1928 17 DNA Homo sapiens 28 gcgcaggttc cgcaggc 17 29 18 DNA Homo sapiens29 gagccactcc acgcaccg 18 30 18 DNA Homo sapiens 30 gagccactcc acgcacgt18 31 19 DNA Homo sapiens 31 cctccaggta ggctctctg 19 32 17 DNA Homosapiens 32 ccgcggagga agcgcca 17 33 21 DNA Homo sapiens 33 ccactccatgaggtatttct t 21 34 19 DNA Homo sapiens 34 ccggagtatt gggacctgc 19 35 18DNA Homo sapiens 35 ccccaggtcg caagccag 18 36 17 DNA Homo sapiens 36cgcacgggcc gcctcca 17 37 18 DNA Homo sapiens 37 gcgccgtgga tagagcaa 1838 17 DNA Homo sapiens 38 gccgcgagtc cgaggac 17 39 19 DNA Homo sapiens39 accggaacac acagatctg 19 40 19 DNA Homo sapiens 40 accgggagacacagatctc 19 41 19 DNA Homo sapiens 41 ggagtattgg gaccggaac 19 42 18 DNAHomo sapiens 42 aacatgaagg cctccgcg 18 43 20 DNA Homo sapiens 43gaccggaaca cacagatctt 20 44 18 DNA Homo sapiens 44 taccgagaga acctgcgc18 45 17 DNA Homo sapiens 45 agcaggaggg gccggaa 17 46 18 DNA Homosapiens 46 ggggagcccc gcttcatt 18 47 19 DNA Homo sapiens 47 cagatctacaaggcccagg 19 48 21 DNA Homo sapiens 48 ccatgaggta tttctacacc g 21 49 20DNA Homo sapiens 49 gaccggaaca cacagatcta 20 50 19 DNA Homo sapiens 50ccgagagagc ctgcgggaa 19 51 19 DNA Homo sapiens 51 accgagagaa cctgcggat19 52 18 DNA Homo sapiens 52 cgccgcgagt ccgagaga 18 53 53 00 54 54 00055 19 DNA Homo sapiens 55 cctccaggta ggctctgtc 19 56 16 DNA Homo sapiens56 gaggaggcgc ccgtcg 16 57 17 DNA Homo sapiens 57 cttgccgtcg taggcgg 1758 19 DNA Homo sapiens 58 atccttgccg tcgtaggct 19 59 20 DNA Homo sapiens59 cgttcagggc gatgtaatct 20 60 18 DNA Homo sapiens 60 cgtgccctccaggtaggt 18 61 18 DNA Homo sapiens 61 gagccactcc acgcactc 18 62 18 DNAHomo sapiens 62 ccaggtatct gcggagcg 18 63 17 DNA Homo sapiens 63ccgcgcgctc cagcgtg 17 64 18 DNA Homo sapiens 64 taccagcgcg ctccagct 1865 20 DNA Homo sapiens 65 gccatacatc ctctggatga 20 66 19 DNA Homosapiens 66 cgtcgcagcc atacatcac 19 67 18 DNA Homo sapiens 67 ctctcagctgctccgcct 18 68 18 DNA Homo sapiens 68 gtcgtaggcg gactggtc 18 69 18 DNAHomo sapiens 69 tcgtaggcgt cctggtgg 18 70 18 DNA Homo sapiens 70ctccaacttg cgctggga 18 71 20 DNA Homo sapiens 71 gtgtgttccg gtcccaatat20 72 19 DNA Homo sapiens 72 cgctctggtt gtagtagcg 19 73 20 DNA Homosapiens 73 gcccacttct ggaaggttct 20 74 18 DNA Homo sapiens 74 ccatacatcgtctgccaa 18 75 17 DNA Homo sapiens 75 gcgcaggttc cgcaggc 17 76 18 DNAHomo sapiens 76 gagccactcc acgcacag 18 77 19 DNA Homo sapiens 77gggtacccag caggacgct 19 78 20 DNA Homo sapiens 78 gagacacaga agtacaagcg20 79 17 DNA Homo sapiens 79 gccgcggtcc aggagct 17 80 18 DNA Homosapiens 80 cgagagagcc tgcggaac 18 81 18 DNA Homo sapiens 81 cgcgagtccgaggatggc 18 82 18 DNA Homo sapiens 82 caggtatctg cggagccc 18 83 21 DNAHomo sapiens 83 ccactcccat gaggtatttc c 21 84 17 DNA Homo sapiens 84gcggcggtcc aggagcg 17 85 19 DNA Homo sapiens 85 cctccaggta ggctctcaa 1986 18 DNA Homo sapiens 86 gcaggttccg caggctct 18 87 18 DNA Homo sapiens87 ggacctgcgg accctgct 18 88 18 DNA Homo sapiens 88 gggagccccg cttcatct18 89 18 DNA Homo sapiens 89 cgccacgagt ccgaggaa 18 90 17 DNA Homosapiens 90 tcccacttgc gctgggt 17 91 17 DNA Homo sapiens 91 ggaggaagcgcccgtcg 17 92 18 DNA Homo sapiens 92 gagcctgcgg accctgct 18 93 18 DNAHomo sapiens 93 cgagtgggcc tgcggaac 18 94 20 DNA Homo sapiens 94gctacgtgga cgacacggct 20 95 19 DNA Homo sapiens 95 ctcggtcagt ctgtgcctt19 96 20 DNA Homo sapiens 96 tctcggtaag tctgtgcctt 20 97 19 DNA Homosapiens 97 tattgggacg aggagacag 19 98 19 DNA Homo sapiens 98 cgtcgtaggcgtactggtc 19 99 18 DNA Homo sapiens 99 cgacgccgcg agccagaa 18 100 18 DNAHomo sapiens 100 gagcccgtcc acgcactc 18 101 20 DNA Homo sapiens 101tcacagactg accgagcgaa 20 102 19 DNA Homo sapiens 102 acggaatgtgaaggcccag 19 103 17 DNA Homo sapiens 103 agcgacgccg cgagcca 17 104 19DNA Homo sapiens 104 ggccggagta ttgggacga 19 105 19 DNA Homo sapiens 105gatagagcag gagaggcct 19 106 20 DNA Homo sapiens 106 tcacagactgaccgagagag 20 107 17 DNA Homo sapiens 107 cccggcccgg cagtgga 17 108 19DNA Homo sapiens 108 gtggatagag caggagggt 19 109 21 DNA Homo sapiens 109agttaatcct tgccgtcgta a 21 110 18 DNA Homo sapiens 110 cactccacgcacgtgcca 18 111 19 DNA Homo sapiens 111 agcgcaggtc ctcgttcaa 19 112 18DNA Homo sapiens 112 ccgtcgtagg cgtgctgt 18 113 19 DNA Homo sapiens 113ccaagagcgc aggtcctct 19 114 21 DNA Homo sapiens 114 acacagatctacaagaccaa c 21 115 20 DNA Homo sapiens 115 ggacccggga gacacagaac 20 11617 DNA Homo sapiens 116 ccccaggtcg cagccac 17 117 18 DNA Homo sapiens117 tctcagctgc tccgccgt 18 118 17 DNA Homo sapiens 118 ctcacgggccgcctcca 17 119 19 DNA Homo sapiens 119 ccgagtgaac ctgcggaaa 19 120 20DNA Homo sapiens 120 tactacaacc agagcgagga 20 121 18 DNA Homo sapiens121 cacgactgac cgagtgag 18 122 18 DNA Homo sapiens 122 agtccaagaggggagccg 18 123 21 DNA Homo sapiens 123 ccactccatg aggtatttct c 21 12419 DNA Homo sapiens 124 cctccaggta ggctctcca 19 125 18 DNA Homo sapiens125 cagcccctcg tgctgcat 18 126 18 DNA Homo sapiens 126 cgcgcgctgcagcgtctt 18 127 18 DNA Homo sapiens 127 cctccaggta ggcttcag 18 128 19DNA Homo sapiens 128 ggtcgcagcc aaacatcca 19 129 20 DNA Homo sapiens 129agcgtctcct tcccattctt 20 130 19 DNA Homo sapiens 130 taccgagagaacctgcgca 19 131 18 DNA Homo sapiens 131 ccttgccgtc gtaggcga 18 132 18DNA Homo sapiens 132 gtcgtaggcg tcctggtc 18 133 20 DNA Homo sapiens 133ccacgtcgca gccatacatt 20 134 17 DNA Homo sapiens 134 gccgcgagtt cgagagg17 135 19 DNA Homo sapiens 135 accgagagaa cctgcggat 19 136 20 DNA Homosapiens 136 gccttcacat tccgtgtgtt 20 137 17 DNA Homo sapiens 137agcccgtcca cgcaccg 17 138 21 DNA Homo sapiens 138 ccactccatg aggtatttcac 21 139 17 DNA Homo sapiens 139 cctgcgcacc gcgctcc 17 140 19 DNA Homosapiens 140 ctctggttgt agtagcgga 19 141 18 DNA Homo sapiens 141gggtaccggc aggacgct 18 142 19 DNA Homo sapiens 142 acggaaagtg aaggcccag19 143 20 DNA Homo sapiens 143 cttcacattc cgtgtctcct 20 144 19 DNA Homosapiens 144 cacgcagttc gtgcggttt 19 145 18 DNA Homo sapiens 145gcagggtccc caggtcca 18 146 20 DNA Homo sapiens 146 gctctggttg tagtagcgga20 147 19 DNA Homo sapiens 147 gacgacacgc tgttcgtga 19 148 18 DNA Homosapiens 148 acgtcgcagc cgtacatg 18 149 19 DNA Homo sapiens 149tccatgaagt atttcacat 19 150 22 DNA Homo sapiens 150 catgaggtatttctacaccg ct 22 151 20 DNA Homo sapiens 151 cactccatga ggtatttcga 20152 20 DNA Homo sapiens 152 cactccatga ggtatttctc 20 153 20 DNA Homosapiens 153 tgaggtattt ctacaccgcc 20 154 19 DNA Homo sapiens 154cctccaggta ggctctgtc 19 155 18 DNA Homo sapiens 155 ctccaggtag gctctccg18 156 18 DNA Homo sapiens 156 ccttgccgtc gtaggcgt 18 157 18 DNA Homosapiens 157 ccttgccgtc gtaggcgg 18 158 18 DNA Homo sapiens 158ccttgccgtc gtaggcga 18 159 18 DNA Homo sapiens 159 ccttgccgtc gtaggcta18 160 19 DNA Homo sapiens 160 tacaagcgcc aggcacaga 19 161 21 DNA Homosapiens 161 atgatgttga cctttccagg g 21 162 23 DNA Homo sapiens 162ttctgtaact tttcatcagt tgc 23 163 19 DNA Homo sapiens 163 tgccaagtggagcacccaa 19 164 19 DNA Homo sapiens 164 gcatcttgct ctgtgcaga 19 165 30DNA Homo sapiens 165 acgcctacga cggcaaggat tacatcgccc 30 166 31 DNA Homosapiens 166 gatggagccg cggtggatag agcaaggagg g 31 167 27 DNA Homosapiens 167 cagttcgtga ggttcgacag cgacgcc 27 168 30 DNA Homo sapiens 168ctgcgcggct actacaacca gagcgaggcc 30 169 20 DNA Homo sapiens 169tccycgcaga ggatttcgtg 20 170 15 DNA Homo sapiens 170 ggagcgcgtg cgggg 15171 17 DNA Homo sapiens 171 acggagcgcg tgcgtct 17 172 19 DNA Homosapiens 172 ggacggagcg cgtgcgtta 19 173 24 DNA Homo sapiens 173gtactcctct cggttataga tgtg 24 174 23 DNA Homo sapiens 174 gatctcttctcggttataga tgc 23 175 17 DNA Homo sapiens 175 gtcgctgtcg aagcgca 17 17616 DNA Homo sapiens 176 tgacgccgct ggggcc 16 177 20 DNA Homo sapiens 177gctgttccag tactcggcgt 20 178 21 DNA Homo sapiens 178 gctgttccagtactcggcgc t 21 179 20 DNA Homo sapiens 179 gctgttccag tactcggcaa 20 18018 DNA Homo sapiens 180 caactccgcc cgggtcct 18 181 21 DNA Homo sapiens181 gaaggacatc ctggagagga a 21 182 21 DNA Homo sapiens 182 ggtcgtgcggagctccaact g 21 183 21 DNA Homo sapiens 183 cactctcctc tgcaggatcc c 21184 20 DNA Homo sapiens 184 ccccmcagca cgtttcttga 20 185 19 DNA Homosapiens 185 ccagcacgtt tcttggagg 19 186 20 DNA Homo sapiens 186mcagcacgtt tcttggagct 20 187 19 DNA Homo sapiens 187 cacgtttcttgcagcagga 19 188 19 DNA Homo sapiens 188 cacgtttctt ggagctgcg 19 189 23DNA Homo sapiens 189 cgtttcttgg agcaggctaa gtg 23 190 23 DNA Homosapiens 190 cgtttcttgg agtactctac ggg 23 191 23 DNA Homo sapiens 191acgtttcttg gagcaggtta aac 23 192 22 DNA Homo sapiens 192 cgtttcctgtggcagcctaa ga 22 193 23 DNA Homo sapiens 193 cgtttcttgg agtactctac gtc23 194 25 DNA Homo sapiens 194 cgtttcctgt ggcagggtaa gtata 25 195 23 DNAHomo sapiens 195 gttatggaag tatctgtcca ggt 23 196 17 DNA Homo sapiens196 cggagcgggt gcggttg 17 197 18 DNA Homo sapiens 197 acggagcgggtgcggttg 18 198 22 DNA Homo sapiens 198 actcctcctg gttatagaag tg 22 19918 DNA Homo sapiens 199 gctgtcgaag cgcaagtc 18 200 19 DNA Homo sapiens200 tcgctgtcga agcgcacga 19 201 18 DNA Homo sapiens 201 gctgtcgaagcgcaggag 18 202 19 DNA Homo sapiens 202 cgctgtcgaa gcgcacgtt 19 203 17DNA Homo sapiens 203 gctgtcgaag cgcacgg 17 204 18 DNA Homo sapiens 204gctgtcgaag cgcacgta 18 205 18 DNA Homo sapiens 205 cgctgtcgta gcgcgcgt18 206 16 DNA Homo sapiens 206 tccgtcaccg cccgga 16 207 18 DNA Homosapiens 207 ggagtaccgg gcggtgag 18 208 19 DNA Homo sapiens 208ctgttccagt actcggcat 19 209 19 DNA Homo sapiens 209 tgttccagta ctcggcgct19 210 19 DNA Homo sapiens 210 ctgttccagg actcggcga 19 211 21 DNA Homosapiens 211 tcaggctgtt ccagtactcc t 21 212 19 DNA Homo sapiens 212cgcgcctgtc ttccaggaa 19 213 19 DNA Homo sapiens 213 cccgctcgtc ttccaggat19 214 18 DNA Homo sapiens 214 caccgcggcc cgcctctg 18 215 15 DNA Homosapiens 215 caccgcggcc cgcgc 15 216 19 DNA Homo sapiens 216 tgcaataggtgtccacctc 19 217 19 DNA Homo sapiens 217 tgcagtaggt gtccaccag 19 218 24DNA Homo sapiens 218 gtgtctgcag taattgtcca cctg 24 219 23 DNA Homosapiens 219 gtgtctgcag taattgtcca ccc 23 220 17 DNA Homo sapiens 220atgtctgcag taggtgc 17 221 23 DNA Homo sapiens 221 ctctccacca acccgtagttgta 23 222 19 DNA Homo sapiens 222 tgcactgtga agctctcac 19 223 20 DNAHomo sapiens 223 ctgcactgtg aagctctcca 20 224 20 DNA Homo sapiens 224ccccgtagtt gtgtctgcaa 20 225 18 DNA Homo sapiens 225 gcagtaggtg tccaccgc18 226 18 DNA Homo sapiens 226 gcaataggtg tccacctc 18 227 20 DNA Homosapiens 227 ccttctggct gttcccagtg 20 228 19 DNA Homo sapiens 228tccttctggc tgttccagg 19 229 19 DNA Homo sapiens 229 acagtgaagc tctccacag19 230 17 DNA Homo sapiens 230 ctccgtcacc gcccgga 17 231 18 DNA Homosapiens 231 ctccgtcacc gcccggta 18 232 21 DNA Homo sapiens 232ctcctcctgg ttatggaact g 21 233 21 DNA Homo sapiens 233 ctcctcctggttatggaagt a 21 234 21 DNA Homo sapiens 234 tcgctgtcga agcgcacgtc g 21235 21 DNA Homo sapiens 235 cgctgtcgaa gcgcaacgga t 21 236 20 DNA Homosapiens 236 cgctgtcgaa gcgcacgtcg 20 237 19 DNA Homo sapiens 237tcgctgtcga agcgcagga 19 238 19 DNA Homo sapiens 238 tcgctgtcga agcgcacga19 239 20 DNA Homo sapiens 239 acgtcgctgt cgaagcgcag 20 240 19 DNA Homosapiens 240 tcaccgcccg gtactccct 19 241 19 DNA Homo sapiens 241ccaagctccg tcaccgcct 19 242 17 DNA Homo sapiens 242 ccgccccagc tccgtcg17 243 20 DNA Homo sapiens 243 gctgttccag tgctccgcag 20 244 20 DNA Homosapiens 244 gctgttccag tgctccgcat 20 245 20 DNA Homo sapiens 245ggctgttcca gtactcagcg 20 246 20 DNA Homo sapiens 246 gctgttccagtactcggcga 20 247 21 DNA Homo sapiens 247 ttctggctgt tccagtactc a 21 24818 DNA Homo sapiens 248 ccgcctctgc tccaggag 18 249 18 DNA Homo sapiens249 ccgcgcctgc tccaggat 18 250 18 DNA Homo sapiens 250 accgcggcgcgcctgtct 18 251 17 DNA Homo sapiens 251 ccgcggcccg cgcctgc 17 252 18 DNAHomo sapiens 252 caccgcggcg cgcctgtt 18 253 17 DNA Homo sapiens 253cacctcggcc cgcctcc 17 254 18 DNA Homo sapiens 254 gtccaccgcg gcgcgcgt 18255 18 DNA Homo sapiens 255 tgtccaccgc ggcccgct 18 256 17 DNA Homosapiens 256 tccaccgcgg cccgcgc 17 257 17 DNA Homo sapiens 257 tccaccgcggcccgctc 17 258 18 DNA Homo sapiens 258 tgtccaccgc ggcccgct 18 259 18 DNAHomo sapiens 259 taggtgtcca ccgcggcg 18 260 19 DNA Homo sapiens 260gcgccacctg tggatgacg 19 261 21 DNA Homo sapiens 261 tctgcagtaattgtccacct g 21 262 21 DNA Homo sapiens 262 gtctgcaata ggtgtccacc t 21263 20 DNA Homo sapiens 263 ctgcagtagt tgtccacccg 20 264 22 DNA Homosapiens 264 ccgtagttgt atctgcagta gt 22 265 22 DNA Homo sapiens 265ccgtagttgt gtctgcagta gt 22 266 23 DNA Homo sapiens 266 cccgtagttgtgtctgcagt aat 23 267 21 DNA Homo sapiens 267 cccgtagttg tgtctgcaca c 21268 21 DNA Homo sapiens 268 cagcacgttt cttggagctg t 21 269 23 DNA Homosapiens 269 ttcttgtggc agcttaagtt tga 23 270 21 DNA Homo sapiens 270gatccccctg aggtgaccgt g 21 271 21 DNA Homo sapiens 271 ctgggcccgggggtcatggc c 21 272 30 DNA Homo sapiens 272 cacgtcgctg tcgaagcgcacgtactcctc 30 273 30 DNA Homo sapiens 273 cacgtcgctg tcgaagcggacgatctcctt 30 274 30 DNA Homo sapiens 274 cacgtcgctg tcgaagcgtgcgtactcctc 30 275 30 DNA Homo sapiens 275 cacgtcgctg tcgaagcgcgcgtactcctc 30 276 30 DNA Homo sapiens 276 cacgtcgctg tcgaagcgcacgtcctcctc 30 277 30 DNA Homo sapiens 277 tggcgtgggc gaggcagggtaacttcttta 30 278 30 DNA Artificial Sequence Description of ArtificialSequenceCapture Oligonucleotide1 278 accgcacccg ctccgtccca ttgaagaaat 30

What is claimed is:
 1. A method for identifying an HLA genotype of asubject, the method comprising: (a) obtaining a sample comprising atemplate nucleic acid from said subject; (b) amplifying said templatenucleic acid with a plurality of HLA allele-specific forward primers andHLA allele-specific reverse primers to form amplification products,wherein said forward primers or reverse primers comprise a detectablelabel; (c) hybridizing said amplification products with a plurality ofHLA locus-specific capture oligonucleotides immobilized on a solid phaseto form a plurality of detectable complexes; and (d) detecting saiddetectable complexes to identify said HLA genotype of said subject.
 2. Amethod for identifying an HLA genotype of a subject, the methodcomprising: (a) obtaining a sample comprising a template nucleic acidfrom said subject; (b) amplifying said template nucleic acid with aplurality of HLA allele-specific forward primers and HLA allele-specificreverse primers to form amplification products, wherein said forwardprimers or reverse primers comprise a detectable label; (c) hybridizingsaid amplification products with a plurality of HLA locus-specificcapture oligonucleotides to form a plurality of detectable complexes;(d) immobilizing said detectable complexes on a solid phase; and (e)detecting said detectable complexes to identify said HLA genotype ofsaid subject.
 3. The method according to claim 1 or 2, wherein saidtemplate nucleic acid is isolated from blood or cord blood.
 4. Themethod according to claim 1 or 2, wherein said template nucleic acid iscDNA or genomic DNA.
 5. The method according to claim 1 or 2, whereinsaid solid phase is a member selected from the group consisting of: abead, a chip, a microtiter plate, a polycarbonate microtiter plate,polystyrene microtiter plate, and a slide.
 6. The method according toclaim 1 or 2, wherein said HLA genotype is a class I HLA genotype. 7.The method according to claim 1 or 2, wherein said HLA allele-specificforward primers and HLA allele-specific reverse primers are selectedfrom the group consisting of: SEQ ID NOS:1-160.
 8. The method accordingto claim 1 or 2, wherein said locus-specific capture oligonucleotidesare selected from the group consisting of: SEQ ID NOS:165-168.
 9. Themethod according to claim 8, wherein said capture oligonucleotidesfurther comprise a 5′ amine group or a 5′(T)₅₋₂₀ oligonucleotidesequence.
 10. The method according to claim 1 or 2, wherein said HLAgenotype is a class II HLA genotype.
 11. The method according to claim 1or 2, wherein said HLA allele-specific forward primers and HLAallele-specific reverse primers are selected from the group consistingof:selected from the group consisting of: SEQ ID NOS: 169-269.
 12. Themethod according to claim 1 or 2, wherein said locus-specific captureoligonucleotides are selected from the group consisting of: SEQ ID NOS:270-275.
 13. The method according to claim 12, wherein said captureoligonucleotides further comprise a 5′ amine group or a 5′(T)₅₋₂₀oligonucleotide sequence.
 14. The method according to claim 1 or 2,wherein said detectable label comprises a member selected from the groupconsisting of: radioactive moiety, a fluorescent moiety, achemiluminescent moiety, an antigen, and a binding protein.
 15. Themethod of claim 14, wherein said fluorescent moiety is fluorescein or5-(2′-aminoethyl) aminonaphtalene-1-sulfonic acid (EDANS).
 16. A methodfor identifying an HLA genotype of a subject, the method comprising: (a)isolating template nucleic acid from a sample from said subject; (b)immobilizing a plurality of HLA allele-specific reverse primers on asolid phase; (c) amplifying said template nucleic acid with a pluralityof HLA allele-specific forward primers and said immobilized reverse HLAallele-specific reverse primers to form amplification products, whereinsaid forward primers comprise a detectable label; and (d) detecting saidamplification products to identify said HLA genotype of said subject.17. The method according to claim 16, wherein said template nucleic acidis cDNA or genomic DNA.
 18. The method according to claim 16, whereinsaid template nucleic acid is isolated from blood or cord blood.
 19. Themethod according to claim 16, wherein said solid phase is a memberselected from the group consisting of: a bead, a chip, a microtiterplate, a polycarbonate microtiter plate, polystyrene microtiter plate,and a slide.
 20. The method according to claim 16, wherein said HLAgenotype is a class I HLA genotype.
 21. The method according to claim16, wherein said HLA allele-specific reverse primers and said HLAallele-specific forward primers are selected from the group consistingof: SEQ ID NOS:1-160.
 22. The method according to claim 16 wherein saidHLA allele-specific reverse primers further comprise a 5′ amine group ora 5′(T)₅₋₂₀ oligonucleotide sequence.
 23. The method according to claim16, wherein said HLA genotype is a class II HLA genotype.
 24. The methodaccording to claim 16, wherein said HLA allele-specific reverse primersand said HLA allele-specific forward primers are selected from the groupconsisting of: SEQ ID NOS: 169-269.
 25. The method according to claim16, wherein said detectable label comprises a member selected from thegroup consisting of: radioactive moiety, a fluorescent moiety, achemiluminescent moiety, an antigen, and a binding protein.
 26. Themethod of claim 25, wherein said fluorescent moiety is fluorescein or5-(2′-aminoethyl) aminonaphtalene-1-sulfonic acid (EDANS).
 27. Themethod of claim 16, wherein said forward primers and said reverseprimers are selected from the group consisting of: SEQ ID NOS:1-160. 28.The method of claim 16, wherein said forward primers and said reverseprimers are selected from the group consisting of: SEQ ID NOS: 169-269.